1. Structural Biology and Molecular Biophysics
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Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone

  1. Abhilash Padavannil
  2. Prithwijit Sarkar
  3. Seung Joong Kim
  4. Tolga Cagatay
  5. Jenny Jiou
  6. Chad A Brautigam
  7. Diana R Tomchick
  8. Andrej Sali
  9. Sheena D'Arcy
  10. Yuh Min Chook  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. University of Texas at Dallas, United States
  3. Korea Advanced Institute of Science and Technology (KAIST), Korea
  4. University of California, San Francisco, United States
  5. University of California, San Francisco, United states
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Cite this article as: eLife 2019;8:e43630 doi: 10.7554/eLife.43630

Abstract

We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome.

https://doi.org/10.7554/eLife.43630.001

eLife digest

Cells contain two meters of DNA which, if left to its own devices, would soon end up in a knot. To keep things organized, the genetic code is wrapped around protein ‘spools’ called histones, meaning it can all fit within a part of the cell known as the nucleus. The cell makes a copy of its DNA every time it divides, and this copy needs a new set of histones to keep it tidy. The machinery required to construct new histones sits outside the nucleus and getting the histones into position in the nucleus can be a challenge. Histones have a positive charge, which helps to keep the negatively charged DNA wound around the spool. Yet without supervision, histones can stick to other charged molecules in the cell and cause blockages.

The proteins responsible for histone transport are called importins. These proteins normally recognize their cargo by molecular patterns called “nuclear localization signals”. These patterns work like a postal address, telling the importin to take the cargo into the nucleus. When they arrive at their destination, another protein called Ran interacts with the importins to release the cargo. Strangely, removing the predicted address pattern from histones does not stop them getting to the nucleus. To find out what was going on, Padavannil et al. solved the three-dimensional structure of an importin bound to a pair of histones via a technique called X-ray crystallography. This made it possible to see how the proteins fit together.

The structure revealed that, rather than interact with the predicted address pattern, the importin wraps around the core of the histones. This blocks the positive charges, stopping the histones sticking to other molecules on their way to the nucleus. The next challenge was to find out how the cell unhooks the histone cargo from the importin when it arrives in the nucleus; with the positive charges covered by the importin, the histones could not stick to the DNA. Yet, something changed when the levels of Ran were high. Rather than unhook the histone, Ran joined the importin-histone complex. This then made it possible for the histones to attach to DNA, helping them to get into position without sticking to the wrong molecules.

These findings form the first step in understanding how the cell transports sticky histones without getting in a knot. The next step is to find out whether these interactions, shown in test tubes, happen in the same way inside living cells.

https://doi.org/10.7554/eLife.43630.002

Introduction

Eukaryotic chromatin is organized into nucleosomes, which are structural and functional units that are composed of 147 base pairs of DNA wrapped around two H3-H4 dimers and two H2A-H2B dimers (Luger et al., 1997). Nucleosomes are assembled in the nucleus during S-phase as new H2A, H2B, H3 and H4 proteins are synthesized in the cytoplasm (Adams and Kamakaka, 1999; Annunziato, 2012; Verreault, 2000). Newly translated histones are folded and assembled into H2A-H2B and H3-H4 dimers, which are then imported into the nucleus for deposition onto replicating chromatin. Despite their small sizes, histones do not diffuse into the nucleus but are transported by nuclear import receptors of the Karyopherin-β family termed importins (Baake et al., 2001; Jäkel et al., 1999; Johnson-Saliba et al., 2000; Mosammaparast et al., 2002b; Mosammaparast et al., 2001; Mühlhäusser et al., 2001).

Importins usually recognize their protein cargos by binding nuclear localization signals (NLSs) in their polypeptide chains. Importins bind nucleoporins to traverse the permeability barrier of the nuclear pore complex (NPC) (reviewed in Chook and Süel, 2011; Cook et al., 2007; Görlich and Kutay, 1999; Izaurralde et al., 1997; Kim et al., 2018; Kosinski et al., 2016; Lin et al., 2016; Soniat and Chook, 2015). The small GTPase Ran controls direction of transport. Binding of cargos and RanGTP to importins is mutually exclusive. In the nucleus, where Ran is kept in the GTP state by guanine nucleotide exchange factor RCC1, importins bind RanGTP with high affinity, resulting in cargo release (Chook and Süel, 2011; Izaurralde et al., 1997; Soniat and Chook, 2015).

Studies in importin-deletion yeast strains identified Kap114 (S. cerevisiae homolog of Importin-9 or Imp9) as the primary H2A-H2B importer, and Kap121 (homolog of Importin-5) and Kap123 (homolog, Importin-4) as secondary importers (Mosammaparast et al., 2002b; Mosammaparast et al., 2001). Pull-down binding from cytosolic HeLa extract and proteomics tracking nuclear-cytoplasmic localization in human cells also identified core histones as Imp9 cargos (Jäkel et al., 2002a; Kimura et al., 2017). The use of multiple backup importin systems is also seen in human cells, as many previous studies have shown that H2A and H2B can bind and be imported into nuclei of digitonin-permeabilized cells by several human importins (such as Importin-β, Karyopherin-β2, Importin-4, Importin-5, Importin-7) in addition to Importin-9 (Baake et al., 2001; Johnson-Saliba et al., 2000; Mosammaparast et al., 2002b; Mosammaparast et al., 2001; Mühlhäusser et al., 2001).

Core histones H2A, H2B, H3 and H4 all contain disordered N-terminal tails followed by small histone-fold domains; H2A also has a disordered C-terminal tail (Luger et al., 1997). The N-terminal tails of histones contain many basic residues, somewhat resembling classical NLS motifs (Blackwell et al., 2007; Ejlassi-Lassallette et al., 2011; Johnson-Saliba et al., 2000; Marchetti et al., 2000; Greiner et al., 2004; Mosammaparast et al., 2001; Moreland et al., 1987). H2A and H2B tails are able to target heterologous proteins into the nucleus (Mosammaparast et al., 2001), but removal of the tails does not abolish localization of H2A-H2B in the nucleus (Thiriet and Hayes, 2001). Furthermore, analysis of seven different importins binding to H3 and H4 tails vs. full-length H3-H4 vs. H3-H4•Asf1 chaperone complex suggested that specificities for importin-binding reside not only in the tail ‘NLSs’ but also in the histone folds and the bound chaperone (Soniat et al., 2016).

Here, we solved the crystal structure of Imp9 bound to the full-length H2A-H2B dimer to understand how histones are recognized for nuclear import. The superhelical Imp9 wraps around the histone dimer. Most of the N-terminal tails of both H2A and H2B are disordered, and only five residues of the H2B tail contact Imp9. Binding of Imp9 blocks DNA and H3-H4 sites on H2A-H2B, and Imp9 prevents H2A-H2B from aggregating on DNA, consistent with a histone chaperone-like activity for Imp9. Unlike other importin-cargo complexes, RanGTP does not dissociate Imp9•H2A-H2B but binds the complex and enhances its dissociation by DNA. The Ran•Imp9•H2A-H2B complex is also able to promote H2A-H2B assembly into nucleosomes. Formation of the Ran•Imp9•H2A-H2B complex appears to modulate importin-histone interactions to facilitate histone deposition to nuclear targets such as the assembling nucleosome.

Results

Structure of the Imp9•H2A-H2B complex

The major nuclear importer for H2A-H2B in S. cerevisiae is Kap114 (Mosammaparast et al., 2002b; Mosammaparast et al., 2001). Imp9, the human homolog of Kap114, was previously shown to bind and import H2A-H2B (Jäkel et al., 2002a; Kimura et al., 2017; Mühlhäusser et al., 2001). We show Imp9-histone interactions in immunoprecipitation from the cytoplasmic fraction of a stable HeLa cell line expressing mCherry-H2B (Figure 1A). We also show by fluorescence microscopy that Imp9 in these cells localizes mostly to the cytoplasm (Figure 1B). Similar cytoplasmic localization of Imp9 was reported in the Human Protein Atlas (Thul et al., 2017; Uhlen et al., 2017). To understand how Imp9 recognizes histones for nuclear import, we solved the crystal structure of human Imp9 bound to full-length X. laevis H2A-H2B (dissociation constant, KD = 30 nM; Table 1 and Figure 1—figure supplement 1) by single wavelength anomalous dispersion to 2.7 Å resolution (Figure 1—source data 1).

Figure 1 with 2 supplements see all
Interactions between Imp9 and H2A-H2B in the cell and crystal structure of the Imp9 •H2A-H2B complex.

(A) Coimmunoprecipitation (CoIP) studies of H2BmCherry from whole cell, cytoplasmic and nuclear fractions of the lysates from HeLa cells stably expressing H2BmCherry, followed by immunoblots with Imp9, Ran, RFP antibodies. PCNA and MAb414 antibodies are used as loading control antibodies. 10 µg of 1.5 mg lysates are analyzed as CoIP input. Blots are representative of three identical experiments. (B) Subcellular localization of Imp9 and Ran in Hela::H2BmCherry cells. HeLa cells were fixed, permeabilized, incubated with affinity-purified rabbit polyclonal Imp9 antibody and mouse monoclonal anti–Ran antibody, and visualized by confocal microscopy. The secondary antibodies were Alexa 488 conjugated anti–rabbit and Alexa 405 conjugated anti-mouse, respectively. The column on the right contains two-color merge images. (C). The crystal structure of human Imp9 (blue) in complex with X. laevis H2A (yellow)-H2B (red).

https://doi.org/10.7554/eLife.43630.003
Table 1
Imp9-H2A-H2B binding affinities by Isothermal Titration Calorimetry.
https://doi.org/10.7554/eLife.43630.007
Binding speciesKD(nM)*ΔH
(kCal/mol)
ΔS
(Cal/mol.K)
ΔG
(kCal/mol)
Imp9 concentration
correction factor
Imp9 + H2A-H2B30
[10, 70]
−10.2
[−10.6, -9.8]
−0.6−10.00.90 [0.88, 0.92]§
0.90 [0.87, 0.93]
0.90 [0.88, 0.92]
Imp9 + H2AΔTail
-H2B
40
[20, 60]
−11.9
[−12.4,–11.5]
−6.7−10.00.83 [0.81,0.84]
0.86 [0.84,0.88]
0.85 [0.83,0.86]
Imp9 + H2A-H2BΔ(1-35)40
[10, 110]
−12.5
[−13.2,–11.9]
−8.5−10.00.87 [0.82, 0.91]
0.89 [0.86, 0.91]
0.87 [0.83, 0.91]
Imp9 + H2AΔTail-H2BΔTail††40
[10, 100]
−11.7
[−12.2,–11.2]
−5.9−9.91.0 [0.98, 1.03]
0.97 [0.92, 1.01]
0.92 [0.88, 0.96]
Imp9ΔH8loop + H2A-H2B10
[1, 20]
−10.1
[−10.4,–9.9]
2.4−10.80.97 [0.96, 0.99]
1.06 [1.05, 1.07]
0.99 [0.98, 1.00]
Imp9ΔH18-H19loop + H2A-H2B450
[350, 600]
7.9
[7.6, 8.3]
56−8.51.12 [1.1, 1.2]
1.16 [1.12, 1.2]
1.15 [1.11, 1.19]
Imp9ΔH19loop + H2A-H2B40
[10, 100]
−11.0
[−11.4,–10.5]
−3.5−9.90.99 [0.98,1.02]
1.00 [0.98,1.03]
1.00 [0.97,1.04]
  1. * The KD value corresponds to a best-fit value obtained from global analysis of each experimental set carried out in triplicate.

    The 68.3% confidence interval for KD determined by global fit analysis of the triplicates in each experimental set.

  2. The 68.3% confidence interval for ΔH determined by global fit analysis of the triplicates in each experimental set.

    § The 68.3% confidence interval for concentration correction factor of Imp9 is determined by local fit analysis of each individual experiment in an experimental set of triplicates.

  3. H2AΔTail – globular domain of H2A (residues 14–119).

    †† H2AΔTail-H2BΔTail - heterodimer of residues 14–119 of H2A with residues 25–123 of H2B.

  4. The following supplement is available for Table 1:Figure 1—figure supplement 1

Imp9 is made up of twenty tandem HEAT repeats, each containing two antiparallel helices A and B that line the convex and concave surfaces of superhelical-shaped protein, respectively (Figure 1C and Figure 1—figure supplement 2A,B). The concave surface of Imp9 is mostly acidic, with a few small basic patches (Figure 1—figure supplement 2B). This charged concave surface of Imp9 wraps around H2A-H2B, burying 1352 Å2 (26% of the H2A-H2B surface) at three distinct interfaces 1–3 (Figure 2A–D and Figure 2—figure supplements 13). The Imp9-bound H2A-H2B has a canonical histone-fold as in nucleosomes (151 Cα atoms aligned, r.m.s.d. 0.505 Å; PDB ID 1AOI) (Luger et al., 1997). In our structure, the N-terminal and C-terminal tails of H2A (residues 1–16, 101–130) and H2B (1-27, 125-126), the first 14 residues of Imp9 and its H19loop (residues 936–996) were not modeled due to missing electron density.

Figure 2 with 3 supplements see all
Imp9 •H2A-H2B binding interfaces.

(A) The Imp9•H2A-H2B structure is oriented as in Figure 1C. The histones H2A (yellow)-H2B (red) are drawn as cartoons. Imp9 (blue) is represented as surface showing three distinct H2A-H2B binding interfaces (dark blue). (B–D). Details of Interface 1 (B), Interface 2 (C) and Interface 3 (D). Intermolecular contacts are shown as dashed lines.

https://doi.org/10.7554/eLife.43630.008

The N- and C-terminal HEAT repeats of Imp9 (Interfaces 1 and 3) clamp the histone-fold domain while the inner surface of central HEAT repeats 7–8 (Interface 2) interacts with a five-residue segment of the H2B N-terminal tail (Figure 2). Interface 1 on Imp9 comprises the loop that follows helix 2B and the last turns of helices 3B, 4B and 5B (Figure 2B, Figure 2—figure supplements 1A, 2A and 3A). Hydrogen-bonding with H2A-H2B residues caps the C-terminal ends of these Imp9 B helices (Figure 2—figure supplement 1D). Of note is the end-to-end capping of the last turn of Imp9 helix 4B by the first turn of histone H2B helix α2. Interface 1 on the histones involves α2-L2-α3 of H2A and α1-L1-α2 of H2B, which constitute a significant portion of the basic DNA-binding surface found in nucleosomes. Although histones and Imp9 surfaces at this interface are electrostatically complementary (Figure 1—figure supplement 2B), interactions also involve many hydrogen bonds, hydrophobic interactions and main chain interactions (Figure 2—figure supplement 1A,D).

Interface 2 involves Imp9 helices 7B, 8B and the H8loop (connects helices 8A to 8B) binding to the short 28KKRRK32 segment of the H2B N-terminal tail (Figure 2C and Figure 2—figure supplements 1B, 2B–C and and 3B). Electron densities for H2B 28KKRRK32 are weak (see Figure 2—figure supplement 2B–C) and atomic displacement parameters (‘B-factors’) for H2B residues 28–32 are also high (>100 Å2), suggesting dynamic interactions. Charged H2B side chains make electrostatic interactions with several acidic Imp9 residues, while the aliphatic part of these basic side chains and their backbone participate in hydrophobic interactions.

Interface 3 involves the last three HEAT repeats of Imp9, specifically the last turn of helix 18A and the short loop that follows, the H18-19loop, the C-terminal half of helix 19A and the first turn of helix 20B (Figure 2D, Figure 2—figure supplements 1C, 2D and 3C). Instead of the typical basic H2A-H2B residues interacting with the acidic Imp9 residues, charges at Interface 3 are reversed (Figure 1—figure supplement 2B). A basic patch formed by the Imp9 H18-19loop and nearby helices complement an acidic surface on the histones formed by residues from H2A helices α2 and αC, and the C-terminal half of H2B that comprises α2-α3-αC. Of note here are salt bridges between Imp9 residue Arg898 and several acidic residues of H2A (Figure 2D). Many hydrophobic contacts are also found at this interface, and several helices (Imp9 H18A, H19A and histone H2B α2) are capped through hydrogen-bonding with partner proteins (Figure 2—figure supplements 1C and 3C).

Distribution of binding energy in the Imp9•H2A-H2B complex

We analyzed the distribution of binding energy of the extensive Imp9-H2A-H2B interface through mutagenesis of the N-terminal histone tails and several long Imp9 loops and determined KDs of the mutants using isothermal titration calorimetry (ITC; Table 1 and Figure 1—figure supplement 1). Imp9 binds full-length H2A-H2B with high affinity (KD = 30 nM). We did not make mutations to Interface 1 because of the many main-chain interactions found there (Figure 2—figure supplement 1D). Interface 2 involves the H8 loop of Imp9 and the N-terminal tail of H2B, both of which are convenient for deletion mutagenesis. Similarly, two long Imp9 loops (H18-19loop and H19loop) in Interface 3 are convenient for deletion mutagenesis.

H2A-H2B mutant assembled with the core of H2A (residues 14–119) and full-length H2B, hence named H2AΔTail-H2B, has similar binding affinity (KD = 40 nM) as full-length H2A-H2B. This result is consistent with structural observations that H2A residues in its N- and C-terminal tails are disordered and likely do not contact Imp9. Removal of the H2B tail (deleting residues 1–35), generating mutant H2A-H2BΔ(1-35), also did not affect binding affinity (KD = 40 nM). This is not surprising given the weak electron density and high B-factors of H2B 28KKRRK32 bound to Imp9 in Interface 2 (Figure 2—figure supplement 2). A H2A-H2B mutant dimer with only the core domain (H2A residues 14–119 complexed with H2B residues 25–123; named H2AΔTail-H2BΔTail) also bind as tightly to Imp9 as the full-length histones (KD = 40 nM). Removal of the Imp9 H8loop (Imp9ΔH8loop), which forms part of the binding site for H2B 28KKRRK32, also did not decrease binding (KD,Imp9ΔH8loop = 10 nM; Table 1, Figure 1—figure supplement 1E). The histone tails thus do not contribute much binding energy for interactions with Imp9.

At Interface 3, the basic H18-19loop of Imp9 contacts the acidic patch of the histones while the nearby H19loop is mostly disordered and its contribution to histone binding is uncertain. Removal of the H18-19loop reduced the affinity 15-fold (KD = 450 nM; Table 1, Figure 1—figure supplement 1F). We note the endothermic binding reaction that occurred upon truncation of this 40-residue loop. This result suggests substantial contribution of Interface three to the total binding energy. Removal of the H19loop did not affect affinity (KD = 40 nM; Table 1, Figure 1—figure supplement 1G), suggesting that this disordered loop does not participate in H2A-H2B binding.

Imp9 functions biochemically like a histone chaperone

A large portion of the Imp9 interface on H2A-H2B overlaps with the DNA-binding and H3-H4-binding interfaces used in nucleosomes (Figure 3A–C). This feature of Imp9 occluding interfaces used in the nucleosome is common to many H2A-H2B histone chaperones of H2A-H2B (Hammond et al., 2017). Imp9 in fact buries more surface area on H2A-H2B (1352 Å2) than well-characterized H2A-H2B chaperones such as Nap1 (387 Å2), Swr1 (488 Å2), Anp32e (533 Å2), Chz1 (906 Å2), Spt16 of FACT (185 Å2) and YL1 (883 Å2) (Hondele et al., 2013; Hong et al., 2014; Kemble et al., 2015; Luger et al., 1997; Mosammaparast et al., 2002a; Obri et al., 2014; Zhou et al., 2008).

Imp9 has structural and biochemical characteristics of a histone chaperone.

(A) Structure of the nucleosome (1AOI): the orientation on the right shows one of the H2A-H2B dimers (in red and yellow) in the same orientation as H2A-H2B shown in the right panel of B. (B) Imp9-bound H2A-H2B (Imp9 not shown) with its Imp9 interface in dark blue. Orientation of H2A-H2B on the left is the same as in Figures 1C and 2A. (C) Surface representations of the H2A-H2B dimer surface (same orientation as in B) showing nucleosomal DNA (red), nucleosomal H3-H4 (green) and Imp9 (blue) binding interfaces. (D-E) Gel-shift assays to probe chaperone activity of Imp9. Increasing concentrations of Imp9 or Nap1 (0.5, 1.0 and 1.5 molar equivalents of H2A-H2B) were added to pre-formed DNA•H2A-H2B complexes, and the mixtures separated on a native gel stained with ethidium bromide to visualize DNA (D) and with Coomassie Blue to visualize protein (E). The two images of the same gel are horizontally aligned. The histone chaperone Nap1 binds H2A-H2B (E, lanes 4–6) leading to the release of free DNA (D, lanes 4–6). Imp9 also releases free DNA (D, lanes 8–10) as it binds H2A-H2B (E, lanes 8–10).

https://doi.org/10.7554/eLife.43630.012

Histone chaperones are a class of functionally, structurally and mechanistically diverse histone-binding proteins that ‘chaperone’ histones to protect them from promiscuous DNA-histone interactions (Elsässer and D'Arcy, 2012; Mattiroli et al., 2015) in many different contexts surrounding the formation of nucleosomes (Laskey et al., 1978). The observation that Imp9 buries more surface area on H2A-H2B than well-characterized histone chaperones raises the question of whether Imp9 might also function as a histone chaperone. This function is manifested biochemically by the protein outcompeting DNA from non-nucleosomal DNA•H2A-H2B complexes (Andrews et al., 2010; Andrews et al., 2008; Hondele et al., 2013; Hong et al., 2014). To test if Imp9 can compete H2A-H2B from DNA like histone chaperone Nap1, we performed native gel-based competition assays. Titration of Nap1 or Imp9 against DNA•H2A-H2B complexes leads to the release of free DNA as Nap1 or Imp9 binds H2A-H2B (Figure 3D,E). These results suggest that Imp9 can act as a histone chaperone by shielding H2A-H2B from promiscuous interactions while it accompanies the histones from the cytoplasm to the nucleus.

RanGTP does not release H2A-H2B but assembles to form RanGTP•Imp9•H2A-H2B

RanGTP generally binds importins with high affinity to dissociate Importin-cargo complexes and release cargos into the nucleus. However, this appears not to be the case with Imp9•H2A-H2B. When increasing concentrations of RanGTP (5–30 molar equivalents S. cerevisiae Ran(1–177/Q71L)) are added to an immobilized MBP-Imp9•H2A-H2B complex, the histones are not released (Figure 4A; controls shown in Figure 4—figure supplement 1A,C). The RanGTP protein used in these experiments is fully active as it easily dissociates a cargo/NLS from Kapβ2 (Figure 4B and Figure 4—figure supplement 1B). In a separate experiment, the Imp9•H2A-H2B complex also remains intact when added to immobilized MBP-RanGTP (Figure 4C). MBP-RanGTP binds to H2A-H2B-bound Imp9 to form what seems to be a heterotetrameric MBP-RanGTP•Imp9•H2A-H2B complex (Figure 4C).

Figure 4 with 6 supplements see all
RanGTP does not release H2A-H2B but forms a RanGTP•Imp9•H2A-H2B complex.

(A) Pull-down binding assay to probe RanGTP (S. cerevisiae Ran(1–179/Q71L)) interactions with the Imp9•H2A-H2B complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) were added to 2.5 μM MBP-Imp9•H2A-H2B that is immobilized on amylose resin. After extensive washing, the bound proteins were visualized by Coomassie-stained SDS-PAGE. Controls are shown in Figure 4—figure supplement 1. (B) Pull-down binding assays to show RanGTP mediated dissociation of the GST-Kapβ2•MBP-PY-NLS complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) were added to 2.5 μM GST-Kapβ2•MBP-PY-NLS (immobilized). After extensive washing, bound proteins were visualized by Coomassie-stained SDS-PAGE. Controls are shown in Figure 4—figure supplement 1. (C) Pull-down binding assay where preformed Imp9•H2A-H2B was added to immobilized MBP-RanGTP. After washing, the bound proteins were visualized by Coomassie-stained SDS-PAGE. (D) EMSA of Imp9 titrated at 0.5–2.5 molar equivalents to constant H2A-H2B. Upward shift of the Imp9 band shows that Imp9 interacts with H2A-H2B. (E) EMSA of Ran titrated at 1–3 molar equivalents to constant Imp9 (lanes 3–6) or Imp9•H2A-H2B (lanes 7–10). Downward shift of the Imp9 band shows that Imp9 interacts with Ran to form Imp9•RanGTP (compare lanes 4–6 to lane 3), while upward shift of the Imp9•H2A-H2B band shows that a heterotetrameric Ran•Imp9•H2A-H2B complex forms (compare lanes 8–10 to lane 7). No Imp9 or Imp9•RanGTP band is present in lanes 8–10 indicating no dissociation of the Imp9•H2A-H2B complex by RanGTP. Proteins inputs for lanes 1–10 are shown in Figure 4—figure supplement 1D. (F) Analytical ultracentrifugation produced sedimentation profiles for Imp9, H2A-H2B, RanGTP, the 1:1 molar ratio mix of Imp9 and H2A-H2B dimer, the 1:1 molar ratio mix of Imp9 and RanGTP, and the 1:1:3 molar ratio mix of Imp9, H2A-H2B dimer and RanGTP. (G) Molecular weights estimated from merged SAXS profiles (MWSAXS) for Imp9, Imp9•H2A-H2B, Imp9•RanGTP, and RanGTP•Imp9•H2A-H2B, compared with molecular weights from the protein sequences (MWseq).

https://doi.org/10.7554/eLife.43630.013

We examined the interactions of Imp9•H2A-H2B with RanGTP in solution using electrophoretic mobility shift assays and size exclusion chromatography. Electrophoretic mobility shift assays (EMSA) show the formation of a 1:1 complex between Imp9 and H2A-H2B (Figure 4D) as well as between Imp9 and RanGTP (Figure 4E, lanes 3–6). A complex containing equimolar amounts of Imp9, H2A-H2B and RanGTP can also form (Figure 4E, lanes 7–10). Size exclusion chromatography of Imp9•H2A-H2B in the presence of excess RanGTP also shows a large complex that contains Imp9, H2A-H2B and Ran (Figure 4—figure supplement 2).

We used analytical ultracentrifugation to rigorously and quantitatively assess the formation of a heterotetrameric RanGTP•Imp9•H2A-H2B complex. We examined individual Imp9, H2A-H2B and RanGTP proteins, equimolar mixes of Imp9+H2A-H2B and Imp9+RanGTP, and a 1:1:3 molar ratio mix of Imp9, H2A-H2B and RanGTP by analytical ultracentrifugation (protein concentrations 3–10 μM; Figure 4F). Sedimentation coefficient values of the individual proteins estimated from the sedimentation velocity experiments are consistent with their molecular weights: Imp9 (3.7S), H2A-H2B (1.3S) and RanGTP (1.4S). The binary complexes of Imp9•H2A-H2B and Imp9•RanGTP are both larger, at 4.3S and 4.2S, respectively. The mixture of Imp9, H2A-H2B and RanGTP gave peaks at 1.4S (excess RanGTP) and 4.6S. The 4.6S assembly is larger than either Imp9•H2A-H2B or Imp9•RanGTP and is likely the quaternary RanGTP•Imp9•H2A-H2B complex.

We also studied Imp9, Imp9•RanGTP, Imp9•H2A-H2B, and RanGTP•Imp9•H2A-H2B (protein concentrations 31–43 μM) by small angle X-ray scattering (SAXS). SAXS profiles for the four Imp9-containing samples were analyzed to calculate radius of gyration (Rg), maximum particle size (Dmax) and pair distribution function (P(r (Figure 4G, Figure 4—figure supplement 3, Figure 4—source datas 1 and 2). The linearity of the Guinier plots confirms a high degree of homogeneity for each of the SAXS samples (Figure 4—figure supplement 3A–D). Molecular weight of the RanGTP•Imp9•H2A-H2B complex was estimated to be 161.1 KDa by using SAXS MOW (Fischer et al., 2010) a value nearly identical to the expected molecular weight of 163.2 kDa from the sequence thus confirming stability of the 4-polypeptide chain RanGTP•Imp9•H2A-H2B complex in solution (Figure 4G).

We compared the Imp9•H2A-H2B structure with the structures of different importins bound to RanGTP, to predict the Ran-binding site on Imp9. In these structures, RanGTP is always sandwiched between N-terminal and either central or C-terminal HEAT repeats of the importins (Figure 4—figure supplement 4). Importin-RanGTP interactions at the first four HEAT repeats of importins (binding Switch 1, Switch two and α3 of RanGTP) appear to be structurally conserved even though the interface on the opposite side of RanGTP involves different central or C-terminal HEAT repeats in different importins (Chook and Blobel, 1999; Kobayashi and Matsuura, 2013; Lee et al., 2005; Tsirkone et al., 2014; Vetter et al., 1999). Structural alignment of HEAT repeats 1–4 of Imp9 with HEAT repeats 1–4 of Importin-β(1-462)•RanGTP (PDB ID 1IBR (Vetter et al., 1999); r.m.s.d. of 152 Cαs in the alignment is 3.27 Å), Kap95•RanGTP (2BKU (Lee et al., 2005) r.m.s.d. of 152 Cαs in the alignment is 3.20 Å), Kapβ2•RanGTP (1QBK (Chook and Blobel, 1999) r.m.s.d. of 152 Cαs in the alignment is 4.02 Å), Kap121•RanGTP (3W3Z (Kobayashi and Matsuura, 2013); r.m.s.d. of 144 Cαs in the alignment is 2.51 Å), Transportin-SR2•RanGTP (4C0Q; (Maertens et al., 2014) r.m.s.d. of 144 Cαs in the alignment is 5.02 Å) and Importin-13•RanGTP (2 × 19 (Bono et al., 2010); r.m.s.d. of 144 Cαs in the alignment is 3.29 Å), and examination of the six structures at a single orientation of Imp9, show that RanGTP binds in very similar orientations to very similar locations at the N-terminus of these importins (Figure 4—figure supplement 4A–F). Examination of the interactions between the N-terminal HEAT repeats of Kap95, Kap121, Importin-β, Importin-13 and Transportin-SR2 with RanGTP, together with the sequence alignment of this region of the importins show positional/structural conservation of many interacting and potentially interacting (in Imp9) residues (Figure 4—figure supplement 5A–G). These analyses suggest that the N-terminal HEAT repeats of Imp9 are likely to be important in binding RanGTP.

Structural alignment of HEAT repeats 1–4 of Imp9 and Kap121•RanGTP allows us to predict the RanGTP binding site at the N-terminus of Imp9 (Figure 4—figure supplement 6A,B). The prediction is supported by an Imp9 mutant with HEAT repeats 1–3 removed that no longer binds RanGTP (Figure 4—figure supplement 6C–E). This likely Ran-binding site at the N-terminus of Imp9 appears separate from but adjacent to the H2A-H2B binding site (Figure 4—figure supplement 6A,B). The GTPase can most likely access Imp9 without dislodging H2A-H2B but proximity of RanGTP to the histones could modulate Imp9-histones interactions especially the kinetics of binding.

RanGTP•Imp9•H2A-H2B is tuned to release histones for nucleosome assembly

We performed native gel-based competition assays to titrate DNA against Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B. DNA is unable to compete H2A-H2B from Imp9•H2A-H2B (Figure 3D–E and Figure 5A, lanes 5–7) but can compete H2A-H2B from RanGTP•Imp9•H2A-H2B to produce Imp9•RanGTP and DNA•H2A-H2B (Figure 5A–B, lanes 8–10). Unlike Imp9, which efficiently displaces DNA from the DNA•H2A-H2B complex (Figure 5C–D, lanes 4–6), Imp9•RanGTP does not displace DNA from the DNA•H2A-H2B complex (Figure 5C–D, lanes 8–10). These results show that the interaction between Imp9 and H2A-H2B is altered by RanGTP.

Figure 5 with 1 supplement see all
RanGTP modulates Imp9-H2A-H2B interactions for H2A-H2B deposition.

(A, B) DNA is titrated at 0.5, 1 and 2 molar equivalents of preformed Imp9•H2A-H2B (equimolar Imp9 and H2A-H2B mixed together) or RanGTP•Imp9•H2A-H2B (equimolar Imp9, H2A-H2B and RanGTP added together). Images of the same native gel, Coomassie stained in (A) and ethidium bromide stained in (B), are aligned for comparison. DNA cannot compete for H2A-H2B from the Imp9•H2A-H2B, leaving free DNA (B, increasing amounts from lanes 5 to 7) and intact Imp9•H2A-H2B (A, lanes 5–7). In contrast, DNA can compete for H2A-H2B from RanGTP•Imp9•H2A-H2B resulting in Imp9•RanGTP complexes (A, lanes 8–10), DNA•H2A-H2B complexes and very little free DNA (B, lanes 8–10). (C, D) Imp9 or Imp9•RanGTP (equimolar Imp9 and RanGTP added together) is titrated at 0.5–1.5 molar equivalents of H2A-H2B (in a DNA•H2A-H2B 1:7 complex). Images of the same native gel, ethidium bromide stained in (C) and Coomassie stained in (D), are aligned for comparison. Imp9 releases free DNA from DNA•H2A-H2B (C, lanes 3–6) and binds histones to form an Imp9•H2A-H2B complex (D, lanes 4–6). By comparison, Imp9•RanGTP releases little free DNA from DNA•H2A-H2B (C, lanes 7–10). (E) The presence of RanGTP and Imp9 facilitates H2A-H2B deposition onto the nucleosome. Nucleosome assembly assay where either H2A-H2B, Nap1•H2A-H2B, Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B is titrated in molar equivalents of 0.5 and 0.75 to tetrasome (TET; 2.5 µM). Nap1 and Imp9•RanGTP can form nucleosomes (NUC) while Imp9 cannot. Coomassie staining in Figure 5—figure supplement 1B. (F) Nucleosome disassembly assay where either Nap1, Imp9 or Imp9•Ran is titrated in molar equivalents of 0.5 and 0.75 to constant nucleosome (NUC; 2.5 µM). Imp9 can disassemble nucleosomes to tetrasomes while Nap1 and Imp9-Ran cannot. Coomassie staining in Figure 5—figure supplement 1C.

https://doi.org/10.7554/eLife.43630.022

We next tested the ability of Imp9 and Imp9•RanGTP to assemble and disassemble nucleosomes (Figure 5E,F). Like Nap1, Imp9 and Imp9•RanGTP do not influence the stability of the tetrasome (Figure 5—figure supplement 1A). To monitor nucleosome assembly, we titrated H2A-H2B alone or with Nap1, Imp9 or Imp9 +RanGTP against tetrasome and assayed the formation of nucleosomes (Figure 5E). Nucleosomes form from H2A-H2B alone or with H2A-H2B and Nap1 or Imp9 +RanGTP (Figure 5E, lanes 4–5 and 8–9) but not with Imp9 alone (Figure 5E, lanes 6–7). Imp9 will bind H2A-H2B preventing its deposition on tetrasomes to make a nucleosome (Figure 5—figure supplement 1B, lanes 6–7). Notably, in the presence of RanGTP, Imp9 is better at promoting H2A-H2B deposition than either Nap1 or no chaperone. To monitor nucleosome disassembly, we titrated Nap1, Imp9, or Imp9 +RanGTP against nucleosomes (Figure 5F). We see that Imp9 can extract H2A-H2B from the nucleosome to produce tetrasome and Imp9•H2A-H2B (Figure 5F, lanes 5–6; Figure 5—figure supplement 1C), while Nap1 and Imp9 +RanGTP have no effect (Figure 5F, lanes 3–4 and 7–8). These data reinforce the chaperone-like activity of Imp9 and show that Ran influences the interaction between Imp9 and H2A-H2B, possibly through an allosteric mechanism as comparative analysis with other importin•RanGTP complexes suggests that the RanGTP binding site does not overlap with the H2A-H2B binding site. The RanGTP binds the Imp9•H2A-H2B complex to modulate importin-histones interactions to facilitate release of the histones for nucleosome assembly.

Discussion

The solenoid-shaped Imp9 wraps around the folded globular domain of the H2A-H2B dimer, leaving most of the N-terminal tails of H2A, H2B and the C-terminal tail of H2A disordered in the complex. Only the 5-residue 28KKRRK32 segment of the H2B tail contacts Imp9 even though weak electron density and high atomic displacement parameters of the H2B tail suggests that these interactions are dynamic. Our structural observations that Imp9 binds mostly to the globular domain of the H2A-H2B are also consistent with the lack of effect in Imp9 binding when either or both histone tails are deleted (Table 1), and with the previously reported nuclear localization of a mutant of H2A-H2B that lacks both its N-terminal tails (Thiriet and Hayes, 2001). However, very weak dynamic/fuzzy long-range electrostatic interactions between Imp9 and histones tails may still exist - we had previously reported very weak and dynamic interactions between an importin-cargo pair by NMR that could not be observed by X-ray crystallography or detected in mutagenesis/ITC experiments (Yoshizawa et al., 2018). Nevertheless, H2A-H2B thus belongs to a small category of nuclear import cargos that mostly use surfaces of folded domains rather than extended linear nuclear import/localization motifs to bind their importins (Aksu et al., 2016; Bono et al., 2010; Cook et al., 2009; Grünwald and Bono, 2011; Grünwald et al., 2013; Matsuura and Stewart, 2004; Okada et al., 2009).

Imp9-binding blocks both the nucleosomal DNA- and H3-H4-binding sites of H2A-H2B in a manner that is reminiscent of histone chaperone-H2A-H2B interactions. Interestingly, the Imp9•H2A-H2B binding interface is much larger than any known complexes of H2A-H2B or H2A.Z/H2B bound to histone chaperones (Elsässer and D'Arcy, 2012; Mattiroli et al., 2015). Imp9 also acts biochemically like a histone chaperone to prevent H2A-H2B from aggregating with DNA in vitro. The ability of Imp9 to structurally sequester H2A-H2B from promiscuous interactions with DNA and its function in trafficking the histones fit with the broadly defined class of histone-binding proteins known as histone chaperones (Elsässer and D'Arcy, 2012; Mattiroli et al., 2015). It is also generally thought that there is little to no free histones in the cell as they are either bound in nucleosomes or by histone chaperones (Elsässer and D'Arcy, 2012; Mattiroli et al., 2015). Imp9 binds H2A-H2B in the cytoplasm, acts as a storage chaperone in the cytoplasm and a nuclear import receptor to take histones through the NPC (Jäkel et al., 2002a; Kimura et al., 2017; Mosammaparast et al., 2002a; Mosammaparast et al., 2001; Mühlhäusser et al., 2001).

Görlich and colleagues proposed in 2002 that negatively charged importins act as chaperones toward positively charged cargo proteins like histones (Jäkel et al., 2002b). Others also suggested importins acting as chaperones (Lusk et al., 2002). We provide structural evidence to support this proposal as the mostly negatively charged Imp9 indeed shields the mostly positively charged histone-fold domain of H2A-H2B, and perhaps also dynamically shields the extended basic histone tails. The ability of Imp9 to chaperone H2A-H2B, however, goes beyond charge shielding. Despite overall charge complementarity, there are only a few salt bridges at the Imp9•H2A-H2B interface, which also employs hydrophobic interactions and hydrogen bonds, many involving main chains of both proteins. Imp9 also shields many hydrophobic patches on H2A-H2B. The interaction further involves a charge reversal where a basic surface at the C-terminal end of Imp9 interacts with the acidic patch of H2A-H2B. The extensive and persistent interactions that allows Imp9 to surround and shield H2A-H2B also differ significantly from the recently revealed chaperoning interactions of another importin, that of Kapβ2 (or Transportin-1) with the Fused in Sarcoma protein (FUS). Kapβ2-FUS interactions are anchored through high affinity binding at the 26-residue PY-NLS linear motif of FUS that then enable weak, distributed and dynamic interactions with multiple mostly intrinsically disordered regions of FUS, to block formation of higher-order FUS assemblies and liquid-liquid phase separation (Yoshizawa et al., 2018).

The way the Imp9 solenoid wraps around H2A-H2B leaves the predicted N-terminal Ran-binding site of Imp9 accessible and ready to bind RanGTP. We showed by pull-down, electrophoretic mobility shift, size exclusion chromatography, analytical ultracentrifugation and SAXS experiments that Imp9 binds both the histones and RanGTP simultaneously and stably, suggesting that unlike most importin-cargo complexes, Imp9•H2A-H2B is unlikely to be dissociated by RanGTP alone upon entering the nucleus. This finding is not without precedence as Pemberton and colleagues previously showed an assembly that contains Kap114, H2A-H2B, RanGTP and the histone chaperone Nap1 (Mosammaparast et al., 2002a). Unlike the Pemberton study, which found the complex containing RanGTP, histones and importin to be intact in the yeast nucleus, we do not detect interactions between Imp9 and H2A-H2B in the nucleus even though that interaction is easily observed in the cytoplasm (Figure 1A,B). Imp9 is likely dissociated from histones soon after the complex enters the nucleus.

We showed that RanGTP changes the interactions between Imp9 and H2A-H2B as it forms the RanGTP•Imp9•H2A-H2B complex. DNA competes effectively with RanGTP•Imp9•H2A-H2B to produce Imp9•RanGTP and DNA•H2A-H2B even though it is unable to extract H2A-H2B from Imp9•H2A-H2B. Furthermore, RanGTP•Imp9•H2A-H2B is better at promoting H2A-H2B deposition to assemble nucleosome than either Nap1•H2A-H2B or no chaperone, while Imp9 alone cannot deposit H2A-H2B. The GTPase in the RanGTP•Imp9•H2A-H2B complex appears to modulate Imp9-H2A-H2B interactions to facilitate histone release and nucleosome assembly. Accessibility of the N-terminal HEAT repeats of Imp9 in the histones complex may allow formation of the RanGTP•Imp9•H2A-H2B complex, but proximity of the Ran and histones binding sites coupled with the flexibility of the HEAT repeats architecture of Imp9 and the propensity for conformational changes likely changed the kinetics of Imp9-histone binding.

Although histones can be deposited by RanGTP•Imp9•H2A-H2B onto DNA or the tetrasome, it remains unclear how H2A-H2B is released from Imp9 in cells. Assembling nucleosomes may release H2A-H2B from RanGTP•Imp9•H2A-H2B or the histones may be passed to another histone chaperone or nucleosome assembly factor as part of a chaperone hand-off cascade in the nucleus. These questions and the one regarding potential additional roles for Imp9 in the cytoplasm are topics for future studies.

Materials and methods

Constructs, protein expression and purification

Wild-type human Imp9 and Imp9 mutants (Imp9ΔH8loop, residues 371–396 replaced with SGSTGGSGS linker; Imp9ΔH18-19loop, residues 890–906 replaced with GSGTGSGSS; Imp9ΔH19loop, residues 941–996 (GGS)12) were cloned into the pGEX-4T3 vector (GE Healthcare, USA) or the pmalE vector (New England BioLabs, Ipswich, MA) modified to contain a TEV cleavage site (Chook and Blobel, 1999; Chook et al., 2002) and express His6-MBP instead of MBP (pHis6-Mal-TEV). Plasmids expressing the X. laevis histones H2A and H2B were a gift from Bing Li, UT Southwestern Medical Center. The construct for mutant H2BΔ(1-35) was PCR-amplified from the wildtype H2B construct and cloned into pET-3A vector (Novagen, USA).

Imp9 and Imp9 mutants were expressed in BL21 (DE3) E.coli cells (induced with 0.5 mM isopropyl-β-d-1-thiogalactoside (IPTG) for 12 hr at 20°C). Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% glycerol and complete protease inhibitors (Roche Applied Science, Mannheim, Germany)) and then lysed with the EmulsiFlex-C5 cell homogenizer (Avestin, Ottawa, Canada). GST-Imp9 was purified using Glutathione Sepharose 4B (GSH; GE Healthcare) and the GST tag was cleaved using Tev protease on the GSH column. Imp9 was further purified by anion exchange chromatography followed by size-exclusion chromatography (Superdex200, GE Healthcare; final buffer – 20 mM HEPES (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 15% glycerol). MBP-Imp9 was purified using amylose resin (NEB) affinity chromatography. The MBP tag was left intact on MBP fusion proteins, which were used for in vitro pull-down binding assays and analysis by size exclusion chromatography.

Wild type and mutant Xenopus histones H2A, H2B proteins were expressed individually in E.coli BL21 DE3 plysS cells, which were lysed by sonication. The lysate centrifuged at 16000 rpm and the washed pellet was resuspended in unfolding buffer (7 M guanidinium HCl, 20 mM Tris HCl, pH 7.5, 10 mM DTT) and dialyzed overnight in SAU-200 buffer (7 M urea, 20 mM sodium acetate, pH 5.2, 200 mM NaCl, 1 mM EDTA, 5 mM β-mercaptoethanol). The unfolded histone protein samples were further purified with cation exchange chromatography in SAU buffer (200–600 mM NaCl) followed by dialysis overnight in cold water. Mutant H2BΔ(1-35) was purified as described above. Mutant proteins H2AΔTail (contains residues 14–119 of H2A) and H2BΔTail (contains residues 25–123 of H2B) used for Isothermal titration calorimetry were obtained from The Histone Source (Colorado, United States).

H2A-H2B was reconstituted by mixing equimolar concentrations of H2A and H2B in unfolding buffer followed by overnight dialysis into refolding buffer (2 M NaCl, 10 mM Tris HCl, 1 mM EDTA, 5 mM β-mercaptoethanol). The dialyzed sample was concentrated and purified using size-exclusion chromatography in refolding buffer (Luger et al., 1997). Mutant histone dimers (H2A- H2AΔTail-H2B, H2A-H2BΔ(1-35) and H2AΔTail-H2BΔTail) were reconstituted and purified as described above for full-length wild type H2A-H2B (Luger et al., 1997).

His-tagged full-length S. cerevisiae Nap1(C200A/C249A/C272A) in pHAT4 vector was expressed in BL21 (DE3) E. coli cells. Nap1 was purified by affinity chromatography using a GE HisTrap SP FF column followed by ion-exchange chromatography using GE Mono Q 10/100 GL column and gel filtration chromatography using GE Superdex-200 16/600 column (20 mM Tris pH 7.5, 300 mM NaCl, 0.5 mM TCEP).

Ran (Gsp1 (1–179, Q71L)) and MBP-Ran were expressed in E.coli BL21 (DE3) cells as His6 –tag proteins (induced with 0.5 mM IPTG for 12 hr at 20°C). Harvested cells were lysed with the EmulsiFlex-C5 cell homogenizer (Avestin, Ottawa, Canada) and the proteins purified by affinity chromatography on Ni-NTA column. Eluted proteins were loaded with GTP, and RanGTP and MBP-RanGTP were further purified by cation exchange chromatography followed by exchanging into buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 4 mM magnesium acetate, 1 mM DTT, 10% glycerol (Chook and Blobel, 1999; Fung et al., 2015).

Imp9•H2A-H2B complex assembly, crystallization, crystal structure determination

Purified Imp9 was mixed with 10-fold molar excess of H2A-H2B in gel filtration buffer (20 mM HEPES (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 15% glycerol). Imp9•H2A-H2B was separated from excess histones by size-exclusion chromatography and concentrated to 18 mg/ml for crystallization. Selenomethionyl-labeled Imp9 was expressed as described previously (Doublié, 1997) and purified as for Imp9. Selenomethionyl-Imp9•H2A-H2B complex was assembled as for the native complex. Initial crystals were obtained by the sitting drop vapor diffusion method from commercial screens (reservoir solution - 40 mM MES pH 6.5, 3 M potassium formate, and 10% glycerol) and were further optimized by the hanging drop vapor diffusion method. Crystals were cryoprotected in reservoir solution that was supplemented with 15% glycerol, and flash frozen in liquid nitrogen. Selenomethionyl-Imp9•H2A-H2B crystals were obtained in the same conditions as native crystals and were prepared similarly for crystallographic data collection.

Imp9•H2A-H2B native crystals diffracted to a minimum Bragg spacing (dmin) of 2.70 Å and exhibited the symmetry of space group P21212 with cell dimensions of a = 127.4 Å, b = 223.3 Å, c = 131.8 Å and contained two heterotrimers per asymmetric unit. All diffraction data were collected at beamline 19-ID (SBC-CAT) at the Advanced Photon Source (Argonne National Laboratory, Argonne, Illinois, USA) and processed in the program HKL-3000 (Minor et al., 2006) with applied corrections for effects resulting from absorption in a crystal and for radiation damage (Borek et al., 2003; Otwinowski et al., 2003), the calculation of an optimal error model, and corrections to compensate the phasing signal for a radiation-induced increase of non-isomorphism within the crystal (Borek et al., 2010; Borek et al., 2013). These corrections were crucial for successful phasing and stable model refinement. Crystals of Imp9•H2A-H2B displayed mildly anisotropic diffraction. To minimize radiation damage and maximize redundancy, native data was collected in two separate scans of 125 degrees for a total of 250 degrees by translating a single crystal in the X-ray beam. Analysis of the self-Patterson function calculated with the native data revealed a significant off-origin peak at approximately (1/2, 1/2, 1/2) and 27% the height of the origin peak, indicating translational pseudosymmetry.

Phases were obtained from a single wavelength anomalous dispersion (SAD) experiment using the selenomethionyl-Imp9•H2A-H2B protein with data to 2.65 Å. Fifty-four selenium sites were located, phases improved and an initial model containing over 50% of all Imp9•H2A-H2B residues was automatically generated in the AutoBuild routine of the Phenix (Adams et al., 2010) program suite. Completion of this model was performed by manual rebuilding in the program Coot (Emsley et al., 2010). Positional and isotropic atomic displacement parameter (ADP) as well as TLS ADP refinement of native Imp9•H2A-H2B with NCS restraints was performed to a resolution of 2.70 Å using the Phenix program suite with a random 2.1% of all data set aside for an Rfree calculation. The final model for Imp9•H2A-H2B (Rwork = 20.9%, Rfree = 24.0%) contained 2275 residues and 356 waters. The relatively high Rwork and Rfree values are likely due to the presence of translational pseudosymmetry. A Ramachandran plot generated with the program MolProbity (Chen et al., 2010) indicated that 97.1% of all protein residues are in the most favored regions and 0.1% in disallowed regions. Illustrations were prepared with PyMOL (Schrodinger LLC, 2015). Data collection and structure refinement statistics are summarized in Figure 1—source data 1.

Quantification of binding affinities by isothermal titration calorimetry (ITC)

Imp9 and mutant Imp9 proteins were expressed and purified as described above. The wild type full-length H2A, H2B and mutant H2BΔ(1-35) proteins were purified as described above. Mutant H2AΔTail and H2BΔTail proteins were obtained from Histone Source (Colorado, USA). H2A-H2B, H2AΔTail -H2B, H2A-H2BΔ(1-35) and H2AΔTail-H2BΔTail heterodimers were reconstituted and purified as described above. Imp9 or mutant Imp9 proteins and H2A-H2B or H2A-H2B mutant dimers were dialyzed in ITC buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM TCEP and 5% glycerol. ITC experiments were carried out using ITC-200 calorimeter (Microcal, LLC, Northampton, MA, USA) at 20°C with 0.035 mM of Imp9 or mutant Imp9 protein in the sample cell and 0.35 mM H2A-H2B or mutant H2A-H2B protein in the syringe. All samples were thoroughly degassed and then centrifuged at 16000 g for 10 min to remove precipitates. 21 injections each of 1.9 μl except for the first (0.5 μl) were sequentially made in each experiment. The injections were mixed at 300 rpm and consecutive injections were separated by 300 s to allow the peak to return to baseline. All experiments were carried out in triplicates. Data were integrated and baseline corrected using NITPIC (Keller et al., 2012). The baseline corrected and integrated data were globally analyzed in SEDPHAT (Houtman et al., 2007; Zhao et al., 2015) using a model considering a single class of binding sites. SVD-reconstructed thermogram provided by NITPIC, the fit-isotherms and the residuals from SEDPHAT were all plotted using GUSSI (Brautigam, 2015). Individual experiments in the triplicate sets are differently color-coded in Figure 1—figure supplement 1A. For error reporting, we used F-statistics and error-surface projection method to calculate the 68.3% confidence intervals of the fitted data (Bevington). The KD (nM), ΔH (kCal/mol), ΔS (Cal/mol.K), ΔG (kCal/mol) and the Imp9 local concentration correction factors for each set of triplicate experiments are reported in the Table 1.

Pull-down binding assays

Pull-down binding assays were performed by immobilizing purified MBP-Imp9 or MBP-RanGTP (S. cerevisiae Gsp1(1–179/Q71L) on amylose resin (New England BioLabs, Ipswich, MA). 40 μl of 100 μM MBP-Imp9 or MBP-RanGTP was immobilized on 200 μl of amylose resin (50% slurry) in binding assay (BA) buffer containing 20 mM HEPES pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT and 15% glycerol. 100 μl of ~20 μM of immobilized MBP-Imp9 resin was incubated with 100 μl of 400 μM of purified H2A-H2B in a total reaction volume of 400 μl for 30 min at 4°C, followed by five washes each with 400 μl BA buffer. 100 μl of ~20 μM of MBP–RanGTP resin were incubated with 100 μl of 50 μM of purified Imp9•H2A-H2B in a total volume of 400 μl for 30 min at 4°C, followed by five washes each with 400 μl BA buffer.

For RanGTP dissociations assays, a gradient of 10 μl, 20 μl, 40 μl or 60 μl of approximately 500 μM purified RanGTP was added to 50 μl of ~20 μM of immobilized MBP-Imp9 that were pre-bound with H2A-H2B, in a total reaction volume of 400 μl. These binding reactions contain 12.5 μM, 25 μM, 50 μM or 75 μM of RanGTP added to 2.5 μM MBP-Imp9•H2A-H2B. Binding was followed by five washes each with 400 μl of the BA buffer. From each of the reactions, 30 μl of beads after final wash was suspended in 30 μl of BA buffer. 10 μl of the bead slurry sample was analyzed on 12% SDS-PAGE gels and stained with Coomassie Blue for visualization. A control experiment involving immobilized GST-Kapβ2, MBP-PY-NLS (PY-NLS of hnRNP A1), and a gradient of 12.5 μM, 25 μM, 50 μM and 75 μM of RanGTP (prepared as described above for the MBP-Imp9•H2A-H2B experiments) was carried out similarly to show that RanGTP dissociates PY-NLS bound to Kapβ2. 2% of the input Ran-GTP added in each of the binding reactions and approximately 2% of flow-through is also shown in the Coomassie-stained SDS-PAGE gels.

Pull-down binding assay to probe Ran binding to Imp9 versus Imp9Δ1–144 were performed by immobilizing GST-Imp9 or GST-Imp9Δ1–144 on Glutathione Sepharose 4B resin (GE Healthcare Life Sciences). 12.5 ml of lysate from 500 ml cell culture (OD600 = 1) pellet of E. coli expressing GST-Imp9 or GST-Imp9Δ1–144 (containing ~8 mg/ml of GST-Imp9 protein) were incubated on 1 ml of 50% Glutathione Sepharose 4B slurry in BA buffer. The GST-Imp9 or GST-Imp9Δ1–144 bound resin was washed five times, each with 6 ml BA buffer, before the binding assay. 200 μl of 50% slurry GST-Imp9 or GST-Imp9Δ1–144 resin (~12 μM proteins) was incubated with 10 μl of ~500 μM RanGTP in a total reaction volume of 400 μl for 30 min at 4°C, followed by five washes (each with 400 μl BA buffer). After washing, 30 μl of 50% beads slurry was suspended in 30 μl BA buffer. 10 μl of the resulting bead slurry sample was analyzed by Coomassie-stained SDS-PAGE. A control experiment using empty GSH sepharose beads and RanGTP was performed as described above.

Size Exclusion Chromatography

The interaction between RanGTP and Imp9•H2A-H2B complex was probed by size exclusion chromatography (SEC). Imp9, RanGTP, H2A-H2B were purified as described above. First, a series of SEC experiments titrating RanGTP was performed. SEC of Imp9 alone (20 μM), RanGTP alone (60 μM), H2A-H2B alone (20 μM), Imp9 +H2A-H2B 1:1 molar ratio (20 μM) with no RanGTP, 0.5, 1, 2 and 3 molar equivalents of RanGTP were performed in buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM TCEP and 8% (v/v) glycerol. The experiments were performed using a Superdex 200 Increase 10/300 GL column. A second series of SEC experiments using 1:1 Imp9 +H2A-H2B (70 μM) and 1:1:1 Imp9, H2A-H2B, and Ran (70 μM) in the same column and the same buffer were performed with higher concentrations of proteins for visualization of proteins in the SEC fractions by Coomassie-stained SDS-PAGE. A third SEC series involves the mutant MBP-Imp9Δ1–144 that does not bind RanGTP and using a different Superdex 200 Increase10/300 GL column with buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT and 10% glycerol.

Analytical ultracentrifugation

The sedimentation coefficients of individual proteins and protein complexes in the mixture were estimated by monitoring their sedimentation properties in a sedimentation velocity experiment carried out in Beckman-Coulter Optima XL-1 Analytical Ultracentrifuge (AUC). The individual proteins and mixtures of proteins were analyzed in AUC buffer containing 20 mM HEPES pH 7.3, 200 mM sodium chloride, 2 mM magnesium chloride, 2 mM TCEP and 8% glycerol (details below). Protein samples (450 µl) and AUC buffer (450 µl) were loaded into a double sector centerpiece and centrifuged in an eight-hole An-50Ti rotor to 50000 rpm at 20°C. The double sectors were monitored for absorbance at 280 nm (A280). A total of 140 scans were collected and the first 80 scans were analyzed. Buffer density, viscosity of the buffer and partial specific volume of the protein was estimated using SEDNTERP (http://www.rasmb.bbri.org/software/windows/sednterp-philo/). Sedimentation coefficient distributions c(s) (normalized for absorption differences) were calculated by least squares boundary modeling of sedimentation velocity data using SEDFIT program (Schuck, 2000). Sedimentation coefficients sw (weighted-average obtained from the integration of c(s) distribution) and frictional ratios f/f0 were obtained by refining the fit data in SEDFIT (Schuck, 2000). For error reporting, F-statistics and Monte-Carlo for integrated weight-average s values were used (Bevington). Data were plotted using GUSSI (Brautigam, 2015).

Individual proteins, Imp9, RanGTP and H2A-H2B, were purified as described above and dialyzed into the AUC buffer before mixing the samples to the final volume of 450 μL for the AUC experiments. Samples for the AUC experiments contain: 1) 450 μL Imp9 alone (3 μM), 2) 450 μL RanGTP alone (10 μM), 3) 450 μL H2A-H2B (10 μM), 4) 3 μM Imp9 + 3 μM RanGTP in a total volume of 450 μL, 5) 3 μM Imp9 +3 μM H2A-H2B in a total volume of 450 μL, 6) 3 μM Imp9 + 3 μM H2A-H2B + 10 μM RanGTP in a total volume of 450 μL. The proteins were mixed overnight before loading into the AUC cell.

Native gel shift assays

Electrophoretic Mobility Shift Assays

One protein component was held constant at 10 µM and the other was titrated. Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 100 min at 150 V at 4°C in 0.5x TBE (40 mM Tris-HCl pH 8.4, 45 mM boric acid, 1 mM EDTA). Gels were stained with Coomassie Blue.

Competition Assays

Nap1, Imp9 or Imp9-Ran (equimolar Imp9 and RanGTP added together without further purification) were titrated (at 0.5, 1.0 and 1.5 molar equivalents of H2A-H2B) against 147 bp Widom 601 DNA mixed with H2A-H2B at 1:7 (1.5 µM:10.5 µM), or 147 bp Widom 601 DNA was titrated against 10.5 μM Nap1, Imp9 or Imp9-Ran (1:1) pre-mixed with an equimolar amount of H2A-H2B. Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 75 min at 150 V at 4°C in 0.5x TBE. Gels were stained with ethidium bromide and then Coomassie Blue.

Nucleosome Assays

Tetrasomes containing H3-H4 and 147 bp Widom 601 DNA were reconstituted as described in Dyer et al (Dyer et al., 2004). To monitor nucleosome assembly, tetrasomes were held constant at 2.5 µM and H2A-H2B or pre-formed complexes of Nap1-H2A-H2B (1:1), Imp9-H2A-H2B (1:1), or Imp9-H2A-H2B-Ran (1:1:1) were titrated. To monitor nucleosome disassembly, Nap1, Imp9, or Imp9-Ran (1:1) complex was titrated against nucleosomes (2.5 µM). Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 75 min at 150 V at 4°C in 0.5x TBE. Gels were stained with ethidium bromide and then Coomassie Blue.

Small angle x-ray scattering

SAXS experiments examining Imp9, Imp9•H2A-H2B, Imp9•RanGTP, and RanGTP•Imp9•H2A-H2B samples were carried out at Beamline 4–2 of the Stanford Synchrotron Radiation Lightsource (SSRL) in the SLAC National Accelerator Laboratory. At SSRL, the beam energy and current were 11 keV and 500 mA, respectively. A silver behenate sample was used to calibrate the q-range and detector distance. Data collection was controlled with Blu-Ice (McPhillips et al., 2002). We used an automatic sample delivery system equipped with a 1.5 mm-diameter thin-wall quartz capillary within which a sample aliquot was oscillated in the X-ray beam to minimize radiation damage(Martel et al., 2012). The sample was placed at 1.7 m from a MX225-HE (Rayonix, USA) CCD detector with a binned pixel size of 292 by 292 μm (Figure 4—source data 1).

All protein samples for SAXS were expressed and purified as described above. Purified Imp9 was exchanged into SAXS buffer (20 mM HEPES pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, and 10% glycerol) by SEC and concentrated to 5 mg/ml (43 µM of Imp9) for SAXS analysis. The Imp9•H2A-H2B was purified as described above and then exchanged into SAXS buffer by SEC and concentrated to 5 mg/ml (35 µM of Imp9•H2A-H2B) for SAXS analysis. To prepare the Imp9•RanGTP complex, previously purified Imp9 was first mixed with 5-fold molar excess of RanGTP for SEC to separate the Imp9•RanGTP complex from excess Ran. This Imp9•RanGTP complex was then buffer exchanged into SAXS buffer in another round of SEC and concentrated to 5 mg/ml (37 µM of Imp9•RanGTP) for SAXS. To prepare the RanGTP•Imp9•H2A-H2B complex, previously purified Imp9•H2AH2B was mixed with 5-fold molar excess of RanGTP in SAXS buffer for SEC to separate RanGTP•Imp9•H2A-H2B from excess RanGTP. Fractions containing RanGTP•Imp9•H2A-H2B were pooled, concentrated and subjected to a second round of SEC in SAXS buffer, after which the complex was concentrated to 5 mg/ml (31 µM of RanGTP•Imp9•H2A-H2B) for SAXS. The 10% glycerol in the SAXS buffer protects the protein samples from radiation damage during X-ray exposure (Kuwamoto et al., 2004) and our early studies show that low glycerol concentrations (5–20%) do not affect protein compaction (Yoshizawa et al., 2018). All solutions were filtered through 0.1 μm membranes (Millipore) to remove any aggregates. The SAXS profiles were collected at protein concentrations ranging from 0.5 to 5.0 mg/ml. 20 one-second exposures were used for each sample and buffer maintained at 15°C. Each of the resulting diffraction images was scaled using the transmitted beam intensity, azimuthally integrated by SASTool (SasTool, 2013) and averaged to obtain fully processed data in the form of intensity versus q [q = 4πsin(θ)/λ, θ = one half of the scattering angle; λ = X ray wavelength]. The buffer SAXS profile was subtracted from a protein SAXS profile. Subsequently, the mean of the lower concentration (0.5–1.5 mg/ml) profiles in the smaller scattering angle region (q < 0.15 Å−1) and the mean of the higher concentration (2.0–5.0 mg/ml) profiles in the wider scattering angle region (q > 0.12 Å−1) were merged to obtain the final experimental SAXS profiles that are free of the concentration-dependent aggregation or polydispersity effect (Kikhney and Svergun, 2015).

The merged SAXS profiles were initially analyzed using the ATSAS package (Petoukhov et al., 2012) to calculate radius of gyration (Rg), maximum particle size (Dmax), and pair distribution function (P(r)) (Figure 4—figure supplement 3 and Figure 4—source datas 1 and 2). The molecular weight (MWSAXS) of each SAXS sample was estimated using SAXS MOW (Fischer et al., 2010) with a threshold of qmax = 0.25–0.3 Å−1 (Figure 4G and Figure 4—source datas 1 and 2).

Co-immunoprecipitation and immunoblotting

HeLa cells expressing H2BmCherry (Ke et al., 2011) (gift from Prof. Hongtao Yu, UT Southwestern). The HeLa Tet-ON cells (Cellosaurus Accession: HeLa Tet-On (CVCL_IY74)) stably expressing H2B-mCherry were originally created (with cell identity confirmation carried out by STR profiling) in Dr. Hongtao Yu’s lab at University of Texas Southwestern Medical Center, Dallas, Texas USA. Mycoplasma negative status of the cell line was confirmed using the LookOut Mycoplasma PCR Detection kit, Sigma MP0035-1KT. The cells were grown to 80% confluency, and total-cell lysate was prepared by suspending the cells in TB buffer containing 20 mM HEPES–KOH pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.1 mM EGTA, 1 mM DTT and protease inhibitor cocktail (Kimura et al., 2017) on ice for 15 min, sonicating three times (5 s pulse, 10 s rest), then centrifuging the lysed cells at 15,000 g for 20 min at 4°C. Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) as per manufacturer’s instruction. Protein concentration was quantitated using the Bradford protein assay kit (BioRad). The RFP-Trap (high quality Red Fluorescent Protein (RFP) binding protein coupled to a monovalent magnetic matrix, ChromoTek GmbH) was incubated with the cell lysates for 2 hr at 4°C. The matrix was first washed with TB buffer supplemented with 200 mM NaCl, and then once with TB buffer supplemented with 150 mM NaCl. The proteins bound to the beads were dissolved in SDS sample buffer for immunoblot analysis.

Cell lysate and protein samples dissolved in SDS sample buffer were separated by SDS–PAGE, and blotted with the indicated antibodies: Rabbit polyclonal antibody against tagRFP (1:1000 dilution, Cat no. AB233, Evrogen), rabbit polyclonal antibody against Imp9 (1:1000 dilution, Cat no. A305-474A-T, Bethyl Laboratories, Inc), mouse monoclonal against Ran (1:2000 dilution, Cat no. 610340, BD Biosciences), mouse monoclonal against Nuclear Pore Complex Proteins Antibody [MAb414] (1:5000 dilution, Cat no. 902903, BioLegend) and mouse monoclonal against PCNA (1:2000 dilution, Cat no. 307901, BioLegend). Goat anti-Rabbit IgG (H + L), HRP-conjugated (1:6000 dilution, Cat no. 31460, Thermo Fisher Scientific) and Goat anti-Mouse IgG (H + L), HRP-conjugated (1:6000 dilution, Cat no. 31430, Thermo Fisher Scientific) were used as the secondary antibodies, and immunoblots were developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat no. 34580, Thermo Fisher Scientific) according to the manufacturer's protocols and followed by detection using a Gel Doc EZ System (Bio-Rad Laboratories, Hercules, CA, USA.

Confocal microscopy imaging

Cells (5 × 104 cells per chamber) were seeded into collagen coated culture coverslip (BD Falcon) The next day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature followed by permeabilization with 0.1% sodium citrate plus 0.1% Triton X-100. The cells were subjected to immunofluorescence staining using rabbit polyclonal antibody against Imp9 (1:250 dilution, Cat no. A305-474A-T, Bethyl Laboratories, Inc) and mouse monoclonal antibody against Ran (1:250 dilution, Cat no. 610340, BD Biosciences), for 2 hr at room temperature. The cells were then washed with cold PBS three times for 1 min each and incubated with Alexa 480-labeled anti-rabbit secondary antibody (1:800) (Invitrogen) and Alexa 405-labeled anti-mouse secondary antibody (1:800) (Invitrogen) at room temperature for 1 hr. Subsequently cells were washed with cold PBS three times for 1 min each and mounted with ProLong Gold Antifade Mountant (Invitrogen).

Image acquisition was performed with a spinning disk confocal microscope system (Nikon-Andor) with a 100 × oil lens and the MetaMorph softwar. Images were acquired from randomly selected fields as a z-stack with step size of 0.1 μm to give a total of 196 slices. For each selected field of view, three images were taken, an Alexa488 (Imp9) image, and Alexa405 (Ran).

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Decision letter

  1. Andrea Musacchio
    Reviewing Editor; Max Planck Institute of Molecular Physiology, Germany
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone" for consideration by eLife. Your article has been reviewed by three reviewers and the evaluation has been overseen by a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: André Hoelz (Reviewer #1).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you will see, the reviewers praise parts of the work and acknowledge the importance of certain achievements. On the other hand, they also identify several important weaknesses, both in the experimental side and in the interpretation of the results. The prevailing view was that the manuscript, at least in its present form, does not meet the standards expected at eLife.

Reviewer #1:

Padavannil et al., present the structure of a histone H2A:H2B pair in complex with its previously identified nuclear transport factor Importin-9 (Imp9). Subsequently, the authors probe the role of Imp9 in nucleosome biogenesis leading to the conclusion that Imp9 acts as a bona fide histone assembly chaperone. The manuscript is well written and easy to follow. However, the figures and the experiments lack the attention to detail that one expects from the Chook laboratory. While the findings are of potential broad interest to the readership of eLife, the biochemical experiments are insufficient to support key conclusions. There are also additional experiments well within the expertise of the Chook laboratory that would increase the impact of this study. The senior author should carefully re-work the figures and revise the text according to the following points:

Figure 1:

The structure is somewhat unexpected and appears carefully done, with the extensive interface between Imp9 and the H2A:H2B histone pair the most striking feature. It is a great example of the plasticity of β-karyopherins and their ability to recognize cargoes by their shape rather than by a linear nuclear localization sequence. Nevertheless, the statistics reported in Figure 1—source data 1 require major revision (see below). The Imp9 schematic shown in panel B does not convey any additional information required to digest the overall structure and should be moved to the supplement. Panels E-G are difficult to digest, particularly when printed. Labels should be restricted to residues shown as sticks to contact forming positions only. Additionally, a broader box view of each interface presented in the supplement using cross-eye stereo, alongside images of representative electron density would be informative. A sequence alignment of Imp9 homologs, highlighting the residues of the 3 identified H2A:H2B binding interfaces should be incorporated in the supplement demonstrating that this extensive 3-dimensional binding interface is evolutionarily conserved.

Figure 2:

Panel 2A: The cartoon representation of H2A:H2B does not provide useful information. Consider replacing this panel with an overview of the assembled nucleosome, highlighting the H2A:H2B regions where Imp9 binding prevents assembly in the context of intact nucleosomes.

Panel 2C: This experiment should be repeated on a continuous native gel to facilitate direct comparison, please consider showing all pull-down and gel shift assays this way. This experiment also lacks an input demonstrating the quality of the protein components used.

Panel 2D: The molecular weight marker is currently not labeled.

Figure 3:

Panel 3A: Unlike other nuclear import complexes, RanGTP is insufficient to release histone H2A:H2B cargos from Imp9. This experiment is at the heart of the manuscripts novelty and requires further experimental support to sufficiently demonstrate Imp9:H2A:H2B:RanGTP tetramer formation. (1) Loading and input gels, as well as bead controls are missing, each individual component should be tested for unspecific MBP resin binding and these controls should be included in the figure. This issue is common across all the pull-downs presented in the entire manuscript and must be addressed in all cases. (2) The current experiment is a single observation, titrating various RanGTP concentrations to super-stoichiometric levels would demonstrate that this complex is resistant to typical cargo release, particularly in the nuclear environment where RanGTP is plentiful.

Panel 3B: This panel presents a gel shift assay that shows that RanGTP sensitizes the Imp9:H2A:H2B:RanGTP import complex to release the histone pair upon recognition by DNA. This experiment should span a single continuous native gel to allow for direct comparison. Given that 3 replicates were performed, quantitation of the total DNA bound to the Imp9:H2A:H2B:RanGTP heterotetramer should be reported. Furthermore, this data contradicts the concluding statement regarding transfer to alternative histone assembly chaperones, as Imp9 appears capable of directly mediating transfer to nucleosomal DNA. Indeed, it is not intuitive why the Imp9:H2A:H2B:RanGTP complex should be dismantled by double-stranded DNA, as this would expose the hydrophobic H2A:H2B surfaces that interact with H3:H4 in the histone octamer. Instead, for me all data points towards a mechanism whereby a RanGTP primed Imp9:H2A:H2B import complex would serve as a direct H2A:H2B donor for nucleosome assembly. This would be easily testable if the Imp9:H2A:H2B:RanGTP import complex would be mixed with various H3:H4:histone chaperone complexes and histone octamer formation assayed either by pull down or by gel filtration analysis. For example, the H3:H4:Rtt106 complex could be used for this experiment and can be made from bacterially expressed proteins (Su et al., 2012). This assay could also be performed in the presence of the Widom 601 DNA to test whether intact nucleosomes can be assembled. A gel filtration assay would also rule out potential artifacts that are caused by aggregated H2A:H2B pairs in these reported assays.

Panel 3C: The sedimentation coefficient X axis of the analytical ultracentrifugation plot extends too far, it would be useful to focus on a smaller range, perhaps 0-7, to allow for easier identification of the different peaks.

Panel 3D: The nucleotide that is bound to Ran should be labeled in the figure. Furthermore, this panel should clearly be labeled with "MODEL" to prevent readers from concluding that the crystal structure of the RanGTP-bound form of the Imp9:H2A:H2B complex has been determined. The model presumes that RanGTP binds Imp9 in a canonical fashion, modeled on the S. cerevisiae Kap121:RanGTP structure, however its binding does not dismantle the import complex.

Panel 3E: The entire SAXS analysis does not build any confidence that the interaction between RanGTP and the Imp9:H2A:H2B complex is modelled correctly. This data can either be omitted entirely from the manuscript or moved to the supplement. Instead, RanGTP binding to the Imp9:H2A:H2B complex should be validated by Imp9 surface mutagenesis, taking advantage of previously determined crystal structures of β-karyopherins bound to RanGTP. A zoomed in view, detailing the presumably conserved binding determinants shared between Imp9 and the previously characterized β-karyopherins in complex with RanGTP should be incorporated into this figure alongside the site-directed mutagenesis analysis. If the model is correct, mutations should be readily identifiable that mutants that abolish the RanGTP interaction. Additionally, the key take home message from this figure is that RanGTP cannot trigger H2A:H2B release but instead alters the import complex to permit handover to DNA. However, the data presented do not sufficiently rule out the possibility that large excess of RanGTP can trigger release, which is the condition in the nucleus. Thus, a gel filtration interaction analysis experiment, preferably coupled to multi-angle light scattering to determine stoichiometries, showing stable RanGTP:Imp9:H2A:H2B complex formation by incubating Imp9:H2A:H2B with equimolar or 10-fold excess of RanGTP prior to injection would demonstrate complex formation and rule out disassembly.

Figure 1—figure supplement 1: The ITC analysis is commendably thorough. However, the titrations conducted in panels D, E and F must be repeated. The injection interval is too short and does not allow the instrument to equilibrate to baseline. Additionally, standard deviations for the dissociation constants should be reported. Have the experiments been carried out multiple times? Also, ΔG, ΔH and ΔS values should be reported.

Figure 1—figure supplement 1B: The surface charge analysis should be moved to a new figure, with the interface presented in an open book format. Bold outlines denoting the location of the 3 interfaces between Imp9 and H2A:H2B are required to orient the reader to the distinct surfaces involved. Additionally, please use and cite APBS for the surface charge calculations.

Figure 1—source data 1:

This is not a standard X-ray crystallography data collection and refinement statistics table. From top to bottom: Cell dimensions should be reported with 1 decimal point (not 127.424 but 127.4), the B-factors should be reported as full numbers (not 52.81 but 53). The resolution is inconsistently reported at 2.7 Å here and 2.65 Å in the methods. Furthermore, a Rmergeof 0 and I/σ(I) of 1 in the highest resolution shell, I am convinced that the high-resolution limit has not been determined correctly. The I/σ(I) reported in the PDB validation report is 1.9 contradicting what is reported in the table. While direct detectors allow the usage of all data to background levels, proper resolution criteria should be reported. Thus, please report the CC1/2 values and use this parameter to properly determine the high-resolution limit. Rmerge should be reported in percent (not 0.085 but 8.5% ). Rwork and Rfree should only be reported for the entire dataset, omit highest resolution shell. As the structure does not contain ligands or ions, the corresponding atomic and B-factor values can be omitted. 356 water molecules modeled into a structure at 2.7 Å resolution appears unusually high, especially with weak or no data in the highest resolution shell. Were waters modeled into noise? Limit the placement of water molecules to a sensible B-factor. The Molprobity and Ramachandran statistics are missing, these are essential quality control values that must be reported in the table. For both datasets the number of total and unique reflections are missing. Finally, the PDB code is not reported, the authors should formally submit the structure to the PDB and include the accession number in the revised manuscript. Within the methods portion for the crystallization and structure determination both the number of molecules in the ASU and how NCS has been treated during refinement should be detailed.

Reviewer #2:

In this study, the authors solved the crystal structure of Importin-9 in complex with the histone H2A-H2B dimer. Importin-9 functions as a nuclear import receptor for various (typically highly basic) substrates. The importin-cargo complex docks to nuclear pore complex (NPC) and is subsequently translocated to nucleoplasmic side, where RanGTP binds to the Importin with high affinity and favors release of cargo into nucleus. The structure of importin-9 complex will be appreciated in the field of nuclear transport research as it is quite different from other known import receptor crystal structures. Apart from solving the structure, the authors examine, from a biophysical perspective, the role of Importin-9 as a histone (H2A-H2B) chaperone.

In general, the structure is a magnificent achievement and the paper is nice. However, the manuscript has several issues that need to be mended:

1) The authors make a strong point in their Discussion section that the histone tails would not be recognized by importin-9, based on the fact that they don't see an electron density for the interaction. Nevertheless, the binding strength drops by substantial factors when either the H2A or the H2B tail is deleted (4.5-fold and 13-fold, respectively). The combined effect of both deletions will cause a 60-fold drop in binding strength. This is certainly not a minor effect. Given that the authors measured the affinities at a higher than physiological ionic strength, the drop will be even greater under import conditions. Dismissing the tails' contribution is therefore a misinterpretation.

2) The authors should provide explanations for the missing electron densities. Obvious ones are: (A) that the precipitant used for crystallization is of very high ionic strength (3M potassium formate) and therefore likely to artificially break otherwise important salt bridges and (B) that the tails might engage in a fuzzy interaction with the importin. This is an established concept describing interactions of disordered regions with a globular protein. In the specific case, there are probably several possibilities for how the basic tails salt-bridge to negatively charged regions of the importin.

3) In the light of the just made points, I regard the comments on the Muhlhausser, (2001) paper (Discussion section) as inappropriate.

4) Likewise, the authors give the impression as if they had contradicted the concept of importins functioning as chaperones for highly basic cargoes (Discussion section). The concept (Jäkel et al., 2002) implied that importins shield histones and ribosomal proteins against aggregation with polyanions and gave an intuitive explanation as to why import signals are not always as short as an SV40 NLS. It is unclear why the authors phrase their data as a contradiction. It would be more appropriate to give proper credit; the importin-chaperone concept should already be introduced in the Introduction.

5) The referencing in the introduction is inappropriately biased. For example, there is no mentioning of the first discovered import pathway for a histone (Jakel, 1999). Likewise, the by now established fact that the RanGTPase system determines the direction of transport is referenced by two reviews of the Chook lab – despite the fact that the RanGTPase gradient concept had been worked out by a set of papers by the Görlich lab (see e.g. Izaurralde el al., 1997). Please amend this and similar problems.

6) I have little faith in the model of the RanGTP/ importin-9 complex. The Chook lab published a similar prediction before, namely a model of how RanGTP binds to the CRM1/ snurportin 1 complex (Dong et al., 2009). The experimental structure of the RanGTP/CRM1/ snurportin complex (Monecke et al., 2009), however, revealed that the model was not correct. I cannot see why a prediction of the RanGTP/ importin-9 prediction would be any better. There is less than 20% sequence identity to the most similar importin/ exportin with a solved RanGTP structure. Furthermore, the mode of RanGTP-binding is only partially conserved amongst the members of the importin β superfamily. Some conservation is evident in the contact regions of HEAT repeats 1-3. The additional contacts, however, are not conserved; they differ widely between the various solved RanGTP complexes, and I cannot see how one could possibly predict new ones in any reliable manner. A validation of the modeled interaction by mutagenesis will also not help because such validation will not assess the most problematic aspects of the model, namely contacts that have not been predicted. The RanGTP/ importin-9 structure model should be omitted because there is no reason to believe that it is reliable.

7) The signal to noise ratio in the ITC experiments is rather poor – resulting in rather broad confidence intervals of the fits. Some plots e.g. Figure 1—figure supplement1D, E, G, H failed to reach saturation. Please supply a substantially improved dataset. Since the impact of ionic interactions and the magnitude of the effects are important for the argument, a dataset at physiological salt should be included. For measuring weak interactions, it will help to raise the concentration of the titrant to 30-50-fold higher than the analyte.

8) Please identify the used H2A and H2B variants by including the relevant UniProt numbers.

9) Figure 1A (upper panel). Please identify the protein bands.

10) Figure 1B, please improve clarity of the cartoon.

11) Figure 1E-G are too crowded to get the information. Please re-label to improve clarity, and please label only what is really needed for argument.

12) Please clarify which form of Ran (wt vs. Q69L, yeast of human) is used for which experiment.

13) Fitting of the models into SAXS data is not very conclusive. How well do alternate coordinate models fit the SAXS data?

14) The term "NLS" is ambiguous and by many (if not most) cell biologists considered to be an importin α-dependent nuclear import signal. I would therefore refrain from calling the importin 9-interacting interface an NLS. The more generic term "import signal" should cause less confusion.

Reviewer #3:

The manuscript by Padavannil and colleagues (team of Yuh Min Chook and collaborators) reports the crystal structure of human Importin 9 in complex with X. laevis histones H2A-H2B to 2.7A resolution. The 20 HEAT repeats of the importin wrap around one H2A-H2B heterodimer, burying a large surface through three surface regions. Some interaction occurs with a C-terminal segment of the N-terminal H2B tail, but otherwise most contacts are mediated between the H2A-H2B core fold and the importin. Next, the authors probe the relevance of specific contacts using isothermal titration calorimetry and deletion mutants. Some limited analysis is used to compare how Nap1 and Imp9 bind H2A-H2B and displace DNA from the H2A-H2B•DNA complex, confirming the ability of Imp9 of shielding histones from inappropriate DNA contacts. Preliminary pulldown experiments between an immobilized MBP-Imp9 fusion constructs and multiple H2A isoforms indicate a broad range of H2A-H2B isoforms interacting with Imp9. Finally, a strong range of biochemical and biophysical assays, including SAXS experiments, indicate that RanGTP binds the Imp9•H2A-H2B complex, rather than leading to the release of the H2A-H2B cargo, similar to what had been observed in pioneering work with the yeast importin and H2A-H2B by Mosammaparast et al., 2002 (which has been duly cited by the authors).

Overall, the reported in the manuscript are of interest to the histone chaperone field. Moreover, the confirmation of the atypical complex between an importin, its cargo and RanGTP simultaneously is of wider molecular interest. The authors also go to a significant level of analysis to confirm that the Imp9-H2A/H2B-RanGTP complex can be formed in vitro. The previous report of a related interaction for the yeast orthologues somewhat dampens the novelty of this specific finding, yet the structure of the Imp9-H2A-H2B complex is important.

Imp9 does not bind the NLS like sequences on the histone tail but covers its surface. This deserves a bit more discussion and a more rigorous analysis, including in ITC assays, as will be suggested below.

Additional experiments to test the existence of the Imp9-H2A-H2B complex in both cytoplasm and nucleus would strengthen the reported interaction at the level of IP in HeLa cell extracts.

ITC data:

- The results appear to have been derived from single measurements, see Figure 1—figure supplement 1A. As can be seen from this example, there are anomalies in data points in the important region of the titration where the heat of injection changes most. This indicates that the data point for full-length H2A-H2B cannot be trusted. Figure 1—figure supplement 1D, E, and F suffer from similar problems. I would urge the authors to conduct these assays in triplicate. In the absence of these data, the comparison between the distinct H2A-H2B constructs appears premature. I encourage the authors also to provide a stoichiometry value, as well as the ΔH and ΔS parameters from the fitting of the results.

- The ΔH18-19 loop deletion construct shows a quite distinct ITC profile, the fitting here was carried out using an endothermic reaction, rather than fitting exotherms. This is not discussed in the manuscript, but ought to briefly be explained.

- Point mutants might also be quite instructive compared to the rather less surgical deletion of entire histone tails and loops, even if their impact to the overall binding energy may be limited.

(Note: considering the aforementioned limitations on the accuracy of the ITC experiments, providing four significant figures appears unjustified)

Pulldown experiment between importin 9 and H2A-H2B constructs:

- Figure 2D lacks an appropriate negative control, such as an unrelated protein, MBP alone and/or an important mutant/distinct isoform that does not bind H2A-H2B.

-The authors claim that the Imp9-H2A-H2B complex is present in the cytoplasm and nucleus and possibly also on chromatin. However, they do not directly show this. Co-IPs in these different cellular fractions would confirm this and would be a nice addition to the paper.

- Additional assays/evidence, such as imaging, showing that RanGTP can bind with Imp9-H2A/H2B in vivo would strengthen the manuscript.

Gel shift assay in Figure 3B:

- As it stands, the current assay is difficult to interpret, as samples are not shown comparable on the same gel. In addition, the conditions that contain Imp9 are different, one is a preformed complex and the other is not. This also makes it difficult to compare activity and make firm conclusions based on this preliminary experiment. This can be readily experimentally addressed.

Figure 4—figure supplement 1:

- There seems to be less H2A/H2B when RanGTP is added. This experiment should be shown on one gel to compare conditions properly. In addition, this referee would suggest adding more RanGTP to test whether H2A-H2B levels decrease further.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone" for consideration by eLife. Your article has been reviewed Detlef Weigel as the Senior Editor, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.

The Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this submission of a manuscript that was previously reviewed at eLife and has now been resubmitted in a revised form, Padavannil and co-authors have adequately addressed the following concerns:

1) The crystallographic table and crystal structure are now of publishable quality.

2) The manuscript text and figures are now largely of publishable quality.

3) The requested nucleosome assembly/disassembly experiments have been carried out.

4) The revised ITC data are of substantially improved and are now of publishable quality.

5) The RanGTP binding site has been mapped to the N-terminal three HEAT repeats of Imp9.

As already pointed out during the first round of review, the manuscript reports a very interesting set of observation and this new submission is further improved. There are, however, some important issues, raised by reviewer 1 and discussed with the others, that require further consideration.

Essential revisions:

We judge that there is not yet sufficient evidence that the RanGTP:Imp9:H2A:H2B complex is stable in solution, raising the question whether the proposed mechanism is correct that RanGTP binding to the Imp9:H2A:H2B complex is required for priming of the import complex that it can serve as a H2A:H2B donor during nucleosome assembly. Therefore, my criticisms of the revised manuscript refer primarily to the insufficient quality of newly included size-exclusion chromatography (SEC) interaction analysis that was introduced to support the formation of a stable RanGTP:Imp9:H2A:H2B tetramer.

Figure 4—figure supplement 1D. We had requested that the authors carry out additional SEC interaction experiments, ideally combined with a multi-angle light scattering analysis, that would clearly establish that RanGTP binding indeed results in the formation of a heterotetrameric RanGTP:Imp9:H2A:H2B complex, rather than triggering the release of the bound H2A:H2B import cargo and the formation of a heterodimeric Imp9:RanGTP complex, as it has been observed in countless other canonical import complex cases. This analysis is at the heart of the novelty of the presented manuscript. While the authors have included such a SEC analysis, the results of the experiment are uninterpretable. Either the quality of the experiment is insufficient, or the experiment is contrary to the author's conclusion.

The two SDS-PAGE gels of the preincubation run fractions of a mixture of Imp9:H2A:H2B with a 4-fold molar excess of RanGTP do not show stochiometric incorporation of RanGTP into the Imp9:H2A:H2B complex. As judged by the gel filtration profile of RanGTP in isolation, RanGTP elutes as two peaks, consistent with its known weak dimerization behavior: a major peak at 18.2 ml and a second much smaller peak at ~14 ml. The second smaller peak is close to the position where the Imp9:H2A:H2B heterotrimer elutes (12.9 ml). While there is a faint RanGTP band in the SDS-PAGE gel of the preincubation run (lanes 13-16), the band is clearly much weaker than the H2A:H2B bands, indicating at best a sub-stochiometric incorporation of RanGTP into the Imp9:H2A:H2B complex. However, it seems that the faint RanGTP band may not be the result of the incorporation of RanGTP into the Imp9:H2A:H2B complex, but is rather the secondary peak of RanGTP co-eluting independently. An SDS-PAGE gel of the RanGTP control injection is essential to distinguish these two possibilities. Moreover, there is a shift of ~0.4 ml in the elution profiles of RanGTP in isolation compared to the preincubation run profile (this is best seen in the blue and brown major peaks of RanGTP at an elution volume of ~18 ml). Such differences are common when experiments are carried out on different gel filtration columns, with different tubing lengths, or on different FPLC instruments. Additionally, the amounts of the injected proteins are unequal in the various injections and the baselines indicate that the column was not properly equilibrated between different injections. To definitively conclude whether RanGTP stoichiometrically incorporates into the Imp9:H2A:H2B complex, a proper SEC analysis needs to be carried out in which the various components are injected subsequently on the same gel filtration setup. High quality SDS-PAGE gels for all runs are essential for the interpretation of the experiment and should be included in the revised version of the manuscript.

Unfortunately, the poor quality of the newly included SEC analysis casts further doubts on the validity of the SAXS analysis of the RanGTP:Imp9:H2A:H2B complex. At a minimum the current SEC analysis seems to indicate that the RanGTP:Imp9:H2A:H2B heterotetramer is non-stochiometric at the tested Imp9:H2A:H2B concentration of 100 μM. The maximum reported concentration of the RanGTP:Imp9:H2A:H2B complex in the SAXS analysis was 30 μM. Because no stochiometric RanGTP:Imp9:H2A:H2B tetramer was observed in SEC analysis at an injected concentration of 100 μM, which is comparable to the SAXS analysis, if one considers that injected samples are typically ~4-fold diluted on a gel filtration column, it seems unlikely that the SAXS analysis was carried out with a monodisperse sample of the RanGTP:Imp9:H2A:H2B heterotetramer. Unfortunately, the methods provide no information how the authors reconstituted the RanGTP:Imp9:H2A:H2B tetramer for their SAXS analysis. Generally, monodispersity and the formation of a stable species is essential for a meaningful SAXS analysis. Therefore, SAXS experiments are typically carried out either directly following elution from a gel filtration column or better yet by directly coupling the gel filtration column to the SAXS cell.

The observed difference of the major sedimentation coefficient peaks in the AUC analysis of the Imp9:H2A:H2B complex in the absence and presence of a 3-fold molar excess of RanGTP seems to indicate heterotetramer formation. However, AUC analyses are typically performed at much lower concentrations than gel filtration interaction analyses (the authors also provide here no detail in the methods about their employed concentrations). While the pull-down and AUC data seem to support the interpretation that RanGTP is indeed incorporated into the Imp9:H2A:H2B complex without releasing the H2A:H2B cargo, I am puzzled why the gel filtration data does not support this conclusion.

The recommendation is that the authors carry out a proper gel filtration interaction analysis as outlined above. As an important control, the SEC interaction analysis should also include the truncated Imp9:H2A:H2B complex that the authors identified to be deficient in RanGTP binding. Additionally, the entire SAXS analysis should be removed from the manuscript and the structural characterization of the RanGTP:Imp9:H2A:H2B complex and the likely associated conformational change upon RanGTP binding should be published separately in a future study. Alternatively, an EM analysis could be included but I do not think this is necessary.

https://doi.org/10.7554/eLife.43630.029

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

[…] Figure 1:

The structure is somewhat unexpected and appears carefully done, with the extensive interface between Imp9 and the H2A:H2B histone pair the most striking feature. It is a great example of the plasticity of β-karyopherins and their ability to recognize cargoes by their shape rather than by a linear nuclear localization sequence. Nevertheless, the statistics reported in Figure 1—source data 1 require major revision (see below).

We performed additional rounds of structure refinement and made major revisions to Figure 1—source data 1.

The Imp9 schematic shown in panel B does not convey any additional information required to digest the overall structure and should be moved to the supplement.

We moved the old Figure 1B to the supplement and enlarged it for clarity. It is now Figure 1—figure supplement 1A.

Panels E-G are difficult to digest, particularly when printed. Labels should be restricted to residues shown as sticks to contact forming positions only. Additionally, a broader box view of each interface presented in the supplement using cross-eye stereo, alongside images of representative electron density would be informative.

To improve clarity of the old Figures 1E-G, we have

1) Enlarged each figure panel.

2) Removed many labels of side chains that are partially hidden, thereby relieving the crowding.

These figure panels are now the new Figure 2B-D.

We also added stereo figures of broader views for each of the three interfaces to the supplement. These new figure panels are now in Figure 2—figure supplement 1A-C.

We also added images showing omit map electron density of loops (loop residues were omitted to calculate the omit maps) in each of the three interfaces. These new figures are now in Figure 2—figure supplement 2A-D.

A sequence alignment of Imp9 homologs, highlighting the residues of the 3 identified H2A:H2B binding interfaces should be incorporated in the supplement demonstrating that this extensive 3-dimensional binding interface is evolutionarily conserved.

We added sequence alignment of Imp9 homologs (human, X. laevis, D. melanogaster and S. cerevisiae) in the Imp9 regions that make up Interfaces 1-3 to the new Figure 2—figure supplement 3A-C. Imp9 residues that contact histones are clearly marked. Residues in the three interfaces are mostly conserved, with Interface 3 being the most conserved. The level of conservation, especially in Interface 1, reflect the use the main chain of many Imp9 residues for interactions with histone residues. We mention this in the figure legend. We also show contacts to Imp9 main chain in Interface 1 in Figure 2—figure supplement 1D. We mention the prevalence of main-chain interactions in manuscript (Results section) where we also refer to Figure 2—figure supplement 1D and Figure 2—figure supplement 3A.

Figure 2:

Panel 2A: The cartoon representation of H2A:H2B does not provide useful information. Consider replacing this panel with an overview of the assembled nucleosome, highlighting the H2A:H2B regions where Imp9 binding prevents assembly in the context of intact nucleosomes.

We replaced the old Figure 2A with two 90° views of the nucleosome (new Figure 3A). The nucleosome on the right has one of its H2A-H2B in a similar orientation as the right view of the Imp9-bound H2A-H2B in the new Figure 3B. This way, readers can easily examine Imp9 contact regions (colored dark blue in Figure 3B) relative to the position of H2A-H2B in the nucleosome (Figure 3A). This pair of cartoon representations nicely complement surface representations of H2A-H2B in Figure 3C that compare Imp9, DNA and H3-H4 interfaces.

Panel 2C: This experiment should be repeated on a continuous native gel to facilitate direct comparison, please consider showing all pull-down and gel shift assays this way. This experiment also lacks an input demonstrating the quality of the protein components used.

The gel in the old Figure 2C was in fact a continuous native gel stained with ethidium bromide. It is now the new Figure 3D. We added labeling and revised the figure legend to make this clear. We added a new Figure 3E, which is the Coomassie-stained native gel of the same experiment in Figure 3D. The gel in Figure 3D shows the DNA while the gel in Figure 3E shows the protein components of the experiment.

Panel 2D: The molecular weight marker is currently not labeled.

We labeled all molecular weight markers in Figure 1A, Figure 4A-C and in all relevant figure supplements.

Figure 3:

Panel 3A: Unlike other nuclear import complexes, RanGTP is insufficient to release histone H2A:H2B cargos from Imp9. This experiment is at the heart of the manuscripts novelty and requires further experimental support to sufficiently demonstrate Imp9:H2A:H2B:RanGTP tetramer formation. (1) Loading and input gels, as well as bead controls are missing, each individual component should be tested for unspecific MBP resin binding and these controls should be included in the figure. This issue is common across all the pull-downs presented in the entire manuscript and must be addressed in all cases.

We added a gel (new Figure 4—figure supplement 1C) that shows controls of Imp9, histones and RanGTP added to MBP immobilized on amylose resin. None of these proteins bind non-specifically to MBP or to the amylose resin.

For the gels in the new Figures 4A and 4B, we show the inputs and also the flowthrough (FT) materials of the pull-down experiments in the new Figure 4—figure supplement 1A, B. Both the inputs and FTs show the large molar equivalents (5-30) of the RanGTP used.

(2) The current experiment is a single observation, titrating various RanGTP concentrations to super-stoichiometric levels would demonstrate that this complex is resistant to typical cargo release, particularly in the nuclear environment where RanGTP is plentiful.

We repeated the experiment by titrating increasing amounts of RanGTP, starting with 5 molar equivalent of RanGTP (to the MBP-Imp9•H2A-H2B) in lane 6 of the new Figure 4A followed by 10 molar equivalents in lane 7, 20 molar equivalents in lane 8 and 30 molar equivalents of RanGTP in lane 9. Each of the four lanes shows similar amounts of H2A-H2B and RanGTP bound to immobilized Imp9. Little H2A-H2B is displaced from the beads in the FT (lanes 24-27 of the gel in the new Figure 4—figure supplement 1A).

The same 5, 10, 20 and 30 molar equivalents of RanGTP were also used in the control experiment of MBP-PY-NLS binding to GST-Kapβ2 (Figure 4—figure supplement 1B). In this case, even the lowest concentration with 5 molar equivalent of RanGTP easily released all MBP-PY-NLS from immobilized Kapβ2.

Panel 3B: This panel presents a gel shift assay that shows that RanGTP sensitizes the Imp9:H2A:H2B:RanGTP import complex to release the histone pair upon recognition by DNA. This experiment should span a single continuous native gel to allow for direct comparison.

We included continuous native gels showing that DNA is better able to compete for histones from Ran•Imp9•H2A-H2B than from Imp9•H2A-H2B (new Figure 5A and B). We show the Coomassie-stained native gel to report on the protein complexes in Figure 5A. The ethidium bromide gel of the same experiment, reporting on DNA•H2A-H2B and free DNA, is shown in Figure 5B.

Given that 3 replicates were performed, quantitation of the total DNA bound to the Imp9:H2A:H2B:RanGTP heterotetramer should be reported.

We are not sure what DNA the reviewer is referring to. The RanGTP•Imp9•H2A-H2B heterotetramer does NOT bind DNA. Lanes 8-10 of Figure 5A and 5B show that bound RanGTP facilitates release of H2A-H2B from Imp9 to DNA. This is in contrast to lanes 57 where it is clear that when H2A-H2B is bound to Imp9 without RanGTP, the histones were not released to DNA.

Furthermore, this data contradicts the concluding statement regarding transfer to alternative histone assembly chaperones, as Imp9 appears capable of directly mediating transfer to nucleosomal DNA. Indeed, it is not intuitive why the Imp9:H2A:H2B:RanGTP complex should be dismantled by double-stranded DNA, as this would expose the hydrophobic H2A:H2B surfaces that interact with H3:H4 in the histone octamer. Instead, for me all data points towards a mechanism whereby a RanGTP primed Imp9:H2A:H2B import complex would serve as a direct H2A:H2B donor for nucleosome assembly. This would be easily testable if the Imp9:H2A:H2B:RanGTP import complex would be mixed with various H3:H4:histone chaperone complexes and histone octamer formation assayed either by pull down or by gel filtration analysis. For example, the H3:H4:Rtt106 complex could be used for this experiment and can be made from bacterially expressed proteins (Su et al., 2012). This assay could also be performed in the presence of the Widom 601 DNA to test whether intact nucleosomes can be assembled. A gel filtration assay would also rule out potential artifacts that are caused by aggregated H2A:H2B pairs in these reported assays.

We took suggestions from reviewer #1 and reviewer #3 to probe the functions of Imp9•H2A-H2B and RanGTP•Imp9•H2A-H2B using established nucleosome assembly assays. We show in the new Figure 5E that RanGTP•Imp9•H2A-H2B enhances the donation of H2A-H2B to preassembled tetrasome to form nucleosome, but Imp9•H2A-H2B is not able to donate H2A-

H2B to the tetrasome. These results are consistent with our suggestion that the Ran•Imp9•H2AH2B complex is likely the relevant complex to deliver H2A-H2B in the nucleus.

We also performed nucleosome disassembly assays (new Figure 5F). Consistent with results of the assembly assays, Imp9 alone is quite efficient at disassembling nucleosome but the presence of RanGTP prevents it from doing so.

Panel 3C: The sedimentation coefficient X axis of the analytical ultracentrifugation plot extends too far, it would be useful to focus on a smaller range, perhaps 0-7, to allow for easier identification of the different peaks.

We changed the extent of the X-axis of the AUC plot to 0 – 6.5 in the new Figure 4F.

Panel 3D: The nucleotide that is bound to Ran should be labeled in the figure. Furthermore, this panel should clearly be labeled with "MODEL" to prevent readers from concluding that the crystal structure of the RanGTP-bound form of the Imp9:H2A:H2B complex has been determined. The model presumes that RanGTP binds Imp9 in a canonical fashion, modeled on the S. cerevisiae Kap121:RanGTP structure, however its binding does not dismantle the import complex.

We considered reviewer #2’s warning that the Ran•Imp9•H2A-H2B cannot be modeled reliably and took the reviewer’s suggestion to remove the model from the main figure. However, we performed extensive comparative structural analysis of the binding of the switch 1 and 2 regions of RanGTP to HEAT repeats 1-4 of Impβ, Kap95, Kapβ2, Kap121, TrnSR and Importin-13 (see Figure 4—figure supplement 3A-F and Figure 4—figure supplement 4A-F). The locations of Ran binding at these N-terminal HEAT repeats are clearly conserved. So are the Ran sites on HEAT repeats 1-4 of exportins (data not shown). The equivalent Ran-binding site at the Nterminus of Imp9 is predicted by superimposing the 1st four HEAT repeats of Imp9•H2A-H2B with the 1st four HEAT repeats of the Kap121•RanGTP complex (Figure 4—figure supplement 3G). The key observation here is that the predicted location of the Ran site at the N-terminus of Imp9 is separate from the nearby H2A-H2B binding site, suggesting that this potential landing pad for Ran is likely available when Imp9 is bound to H2A-H2B.

Panel 3E: The entire SAXS analysis does not build any confidence that the interaction between RanGTP and the Imp9:H2A:H2B complex is modelled correctly. This data can either be omitted entirely from the manuscript or moved to the supplement.

We removed the SAXS model. But, independent of the SAXS model, the MW weights of the complexes obtained from SAXS experiments show that RanGTP is indeed bound to Imp9•H2AH2B – we kept this data (upper portion of old Figure 3E) as the new Figure 4G.

Instead, RanGTP binding to the Imp9:H2A:H2B complex should be validated by Imp9 surface mutagenesis, taking advantage of previously determined crystal structures of β-karyopherins bound to RanGTP.

We show sequence conservation within the first four HEAT repeats of 6 different importins in a sequence alignment in Figure 4—figure supplement 4G. HEAT repeats 1-4 of the 7 importins are of much more conserved (mean pairwise sequence identity of 20%) than for all the HEAT repeats (mean pairwise sequence identity of 17%). It is also clear in Figure 4—figure supplement 3A-F that the locations of the Ran binding site at the N-terminal HEAT repeats of Impβ, Kap95, Kapβ2, Kap121, Trn-SR2 and Importin-13 are also very conserved.

Examination of details in the Ran binding sites (Figure 4—figure supplement 4A-E) and the sequence alignment of HEAT repeats 1-4 of the importins (Figure 4—figure supplement 4G) show that conservation of contact residues is more structural/positional rather than of the sequence. This is explained in detail in the figure legend:

“G. Sequence alignment of residues in HEAT repeats 1-4 of Imp9, Kap95, Kap121, Importin-β, Importin-13 and Transportin-SR2. Importin positions with identical amino acids are shaded red, and those with conserved amino acids are shown in boxes. […] The majority of Imp9 side chains in the most common/structurally conserved Ran contact sites (marked with black circles) are the same as or have similar chemical characteristics as at least one of the five other importin side chains in that same position, supporting the prediction that RanGTP will likely contact Imp9 at the same location as shown in A-E, on the B-helices of HEAT repeats 1-4.”

From this analysis, one wonders if the 8 most common Ran contact positions that also show sequence conservation are the ones that contribute the most to binding energy. However, this had not been previously tested for any of the previously characterized importin-ran complexes and we feel it is not wise to test these 8 sites on Imp9, without support from an Imp9•RanGTP structure. The only previously reported Importin mutant that decreased Ran binding is an N-terminal truncation mutant of Impβ (Kutay et al., 1997). We made a similar deletion mutant by removing residues 1-144 (HEAT repeats 1-3) of Imp9. Imp9Δ1-144 shows decreased binding to RanGTP (Figure 4—figure supplement 3H).

A zoomed in view, detailing the presumably conserved binding determinants shared between Imp9 and the previously characterized β-karyopherins in complex with RanGTP should be incorporated into this figure alongside the site-directed mutagenesis analysis. If the model is correct, mutations shoud be readily identifiable that mutants that abolish the RanGTP interaction.

We show in Figure 4—figure supplement 3A-F the common location of RanGTP binding at the N-termini (HEATs 1-4) of six different Importins. We compared these sites with the equivalent site on Imp9, which was predicted by structurally aligning HEAT repeats 1-4 of the Kap121•RanGTP complex with Imp9 (Figure 4—figure supplement 3G). In the figure that shows this structural alignment, we do not show the C-terminal half of the Imp9•H2A-H2B structure so as not to imply how that half of Imp9 might interact with RanGTP; we do not know the extent of how the superhelix of Imp9 might rearrange upon binding RanGTP. In Figure 4—figure supplement 3A-F, we observed that the superhelices/solenoids of different Importins adopt variable pitches thereby placing different parts of the C-terminal halves of these Importins to interact with the α-4 helix and basic patch of RanGTP.

We show zoomed-in views of interactions at the Ran-binding sites of several importins in the new Figure 4—figure supplement 4A-E. We also show the side chains in the equivalent site on Imp9 (new Figure 4—figure supplement 4F).

Additionally, the key take home message from this figure is that RanGTP cannot trigger H2A:H2B release but instead alters the import complex to permit handover to DNA. However, the data presented do not sufficiently rule out the possibility that large excess of RanGTP can trigger release, which is the condition in the nucleus. Thus, a gel filtration interaction analysis experiment, preferably coupled to multi-angle light scattering to determine stoichiometries, showing stable RanGTP:Imp9:H2A:H2B complex formation by incubating Imp9:H2A:H2B with equimolar or 10-fold excess of RanGTP prior to injection would demonstrate complex formation and rule out disassembly.

We show gel filtration analysis of a large excess of RanGTP (12 molar equivalent of Ran to Imp9•H2A-H2B) added to Imp9•H2A-H2B in Figure 4—figure supplement 1D. All H2A-H2B elute in the peak at 12.8 ml along with RanGTP and Imp9. No histones are observed at higher elution volume, suggesting no dissociation from Imp9 by RanGTP.

We feel strongly that sedimentation velocity and SAXS analyses are far more rigorous compared to gel filtration for determining if RanGTP indeed forms a complex with Imp9•H2AH2B and for probing the molecular weight of the resulting assembly. The gel filtration data serves as the third mode of support (in addition to AUC and SAXS) to the two pull-down binding assays in Figure 4A and 4C to show formation of the heterotetrameric complex. We also added native gel data showing that a larger complex is formed in solution when RanGTP is added to Imp9•H2A-H2B (new Figure 4D and 4E). This data is yet another (the fourth one) independent support for the formation of a stable RanGTP•Imp9•H2A-H2B heterotetramer.

Figure 1—figure supplement 1: The ITC analysis is commendably thorough. However, the titrations conducted in panels D, E and F must be repeated. The injection interval is too short and does not allow the instrument to equilibrate to baseline.

We fixed the problem with this figure. We took all three reviewers’ concerns about our old ITC data seriously and repeated all ITC experiments. The new ITC experiments were performed more rigorously with much higher quality proteins (gels, gel filtration traces and mass spectrometry data shown in this response document) in buffers with 150 mM NaCl, and the data are consistently cleaner. We used single batches of the different proteins to decrease variations that may arise from different preparations of protein reagents. Every set of interaction was evaluated in triplicate experiments. The new data are summarized in the new Table 1 and the thermograms, traces and fits for the triplicate experiments shown in Figure 1—source data 1.

Additionally, standard deviations for the dissociation constants should be reported. Have the experiments been carried out multiple times? Also, ΔG, ΔH and ΔS values should be reported.

We provide something far better than standard deviations – we provide the 68.3% confidence interval for KD and for ΔH determined by global fit analysis of the triplicates in each experimental set. We added ΔH, ΔS and ΔG, values to the new Table 1.

Figure 1—figure supplement 1B: The surface charge analysis should be moved to a new figure, with the interface presented in an open book format. Bold outlines denoting the location of the 3 interfaces between Imp9 and H2A:H2B are required to orient the reader to the distinct surfaces involved. Additionally, please use and cite APBS for the surface charge calculations.

We added open-book views of each of the surface views of Imp9 and H2A-H2B in the figure. These new views are now shown in the bottom panel of the new Figure 1—figure supplement 1B.

Figure 1—source data 1:

This is not a standard X-ray crystallography data collection and refinement statistics table. From top to bottom:

We performed additional rounds of structure refinement. A revised and improved crystallographic statistics table now named Figure 1—source data 1, a PDB validation report and a new methods description for the X-ray crystallographic studies (Materials and methods section) have been provided.

Cell dimensions should be reported with 1 decimal point (not 127.424 but 127.4), the B-factors should be reported as full numbers (not 52.81 but 53).

As the PDB reports unit cell parameters and B-factors to two decimal places (please see the revised PDB validation report), we decided to follow the PDB standard format.

The resolution is inconsistently reported at 2.7 Å here and 2.65 Å in the methods.

Due to low percentage completeness for the resolution shell 2.7 – 2.65 Å, we adjusted the high-resolution limit of the data scaling and the final model refinement to 2.70 Å, which caused no observable change in the electron density map or refinement statistics. Please see the revised Figure 1—source data 1 and the Materials and methods section.

Furthermore, a Rmerge of 0 and I/σ(I) of 1 in the highest resolution shell, I am convinced that the high-resolution limit has not been determined correctly. The I/σ(I) reported in the PDB validation report is 1.9 contradicting what is reported in the table. While direct detectors allow the usage of all data to background levels, proper resolution criteria should be reported. Thus, please report the CC1/2 values and use this parameter to properly determine the high-resolution limit.

The <I/σ(I)> as reported in the PDB validation report (1.8) for the last resolution shell is calculated from intensities estimated from amplitudes (i.e., the structure factors in the mmcif file from the final round of refinement). The <I/σ(I)> as reported in our table (1.0) comes directly from the scaling logfile as output by HKL-3000, and is calculated from the actual intensities. We are not surprised that there is a slight difference between the two values, given the different sources of the intensity values for the calculation.

This X-ray data was collected on a standard CCD detector, an ADSC Quantum315R detector, and not a direct detector. Due to the very high multiplicity of the native data set (10.1 overall, 8.2 for the highest resolution shell), the Rmerge value is quite high, but the precision-indicating merging R(factor) values (i.e., the Rpim) are quite reasonable, as the Rpim describes the precision of the averaged measurement (see Weiss, (2001)). The CC1/2 value for the highest resolution shell is above 0.50, a generally accepted cutoff value for high resolution data shells.

Rmerge should be reported in percent (not 0.085 but 8.5% ). Rwork and Rfree should only be reported for the entire dataset, omit highest resolution shell.

This has been fixed, see the revised Figure 1—source data 1. As the PDB requests these statistics for the overall dataset and the highest resolution shell during the deposition process and records these values in the header of the PDB deposition, we are following the PDB standard format and reporting those statistics in Figure 1—source data 1.

As the structure does not contain ligands or ions, the corresponding atomic and B-factor values can be omitted. 356 water molecules modeled into a structure at 2.7 Å resolution appears unusually high, especially with weak or no data in the highest resolution shell. Were waters modeled into noise? Limit the placement of water molecules to a sensible B-factor.

The ratio of total number of protein residues/water molecules is 2,275/356 = 6.4. A ratio of one water molecule to approximately 6 amino acids is not totally unreasonable for a 2.70 Å map, especially given that the mean refined B-factor for the waters is 36.1 Å2 (and the maximum value is ~58 Å2, which corresponds well with the mean refined B-factors for the Imp9 molecule). The |2Fo – Fc| electron density map, contoured at the 1 r.m.s. level, was visually inspected to ensure that all modeled waters displayed significant electron density associated with each water, and that the modeled waters made appropriate hydrogen bond donor/acceptor interactions with the protein. These modeled waters are primarily in the first hydration shell of the Imp9 protein (chains A and D), which is the best-ordered portion of the model (as evidenced by the lower B-factor values).

The Molprobity and Ramachandran statistics are missing, these are essential quality control values that must be reported in the table. For both datasets the number of total and unique reflections are missing.

We added the requested, see revised Figure 1—source data 1.

Finally, the PDB code is not reported, the authors should formally submit the structure to the PDB and include the accession number in the revised manuscript.

This has been added, see revised Figure 1—source data 1. The PDB ID is 6N1Z.

Within the methods portion for the crystallization and structure determination both the number of molecules in the ASU and how NCS has been treated during refinement should be detailed.

This has been added, see revised Materials and methods section.

Reviewer #2:

[…] 1) The authors make a strong point in their Discussion section that the histone tails would not be recognized by importin-9, based on the fact that they don't see an electron density for the interaction. Nevertheless, the binding strength drops by substantial factors when either the H2A or the H2B tail is deleted (4.5-fold and 13-fold, respectively). The combined effect of both deletions will cause a 60-fold drop in binding strength. This is certainly not a minor effect. Given that the authors measured the affinities at a higher than physiological ionic strength, the drop will be even greater under import conditions. Dismissing the tails' contribution is therefore a misinterpretation.

All three reviewers had substantial concerns about our previous ITC data. We took their concerns seriously and repeated all ITC experiments using single batches of each of the different proteins to decrease variations that might arise from different preparations of protein reagents. Every set of the interactions was evaluated in triplicate. The new data are summarized in the new Table 1 and the thermograms, traces and fits for the triplicate experiments are shown in Figure 1—figure supplement 1.

The new ITC experiments were performed much more rigorously with much higher quality proteins (gels, gel filtration traces and mass spectrometry data shown in this response document) in buffers with 150 mM NaCl, and the data are consistently cleaner. We now see that removal of the H2A tail (H2AΔTail-H2B) or H2B tail (H2A-H2BΔ(1-35)) or removal of both N-terminal tails (H2AΔTail-H2BΔTail) did not affect binding affinity for Imp9. Careful examination of our further refined final structure revealed weak electron density and very high B-factors (>100 Å2) for the short segment of the H2B tail (residues 28-32) that contacts Imp9 at Interface 2 (described in the Results section and shown in Figure 2—figure supplement 2B and 2C). This structural observation is consistent with the mutagenesis/ITC results showing that neither H2A nor H2B tail contributes much to binding Imp9.

2) The authors should provide explanations for the missing electron densities. Obvious ones are: (A) that the precipitant used for crystallization is of very high ionic strength (3M potassium formate) and therefore likely to artificially break otherwise important salt bridges and (B) that the tails might engage in a fuzzy interaction with the importin. This is an established concept describing interactions of disordered regions with a globular protein. In the specific case, there are probably several possibilities for how the basic tails salt-bridge to negatively charged regions of the importin.

Our new ITC data show internal consistency. The data is also consistent with the absent electron density for most of the H2A and H2B N-terminal tails and the very weak electron densities (and very high B-factors) of residues 28-32 of the H2B tail. The H2A and H2B tails do not contribute much binding energy to interactions with Imp9 in solution at physiological ionic strength (ITC performed in buffer with 150 mM NaCl).

It is formally possible that the very high ionic strength of the crystallization condition may artificially prevent dynamic/fuzzy electrostatic interactions between Imp9 and histones in the crystal. But, this is not consistent with our mutagenesis and ITC results. That being said, we did detect very weak and dynamic interactions between an Importin and cargo by NMR that could not be observed by X-ray crystallography or detected in mutagenesis/ITC experiments (Kapβ2 and cargo FUS; Yoshizawa et al., 2018). Therefore, we added discussion(Discussion section) suggesting that very weak dynamic/fuzzy long-range electrostatic interactions between Imp9 and histones tails cannot be completely discounted based on the absence of electron density and the lack of effects observed in mutagenesis/ITC studies.

3) In the light of the just made points, I regard the comments on the Muhlhausser, (2001) paper (Discussion section) as inappropriate.

We assume the reviewer was referring to the first sentence in the previous Discussion section, which referenced the Mosammaparast et al., (2001) paper and not the Muhlhausser (2001) paper. We made extensive revisions to the first paragraph of Discussion section and no longer refer to either the Muhlhausser et al., or the Mosammaparast et al., papers.

That new paragraph now reads:

“The solenoid-shaped Imp9 wraps around the folded globular domain of the H2A-H2B dimer, leaving most of the N-terminal tails of H2A, H2B and the C-terminal tail of H2A disordered in the complex. […] Nevertheless, the H2A-H2B dimer thus belongs to a small category of nuclear import cargos that use surfaces of folded domains rather than extended linear nuclear import/localization motifs to bind their importins (Aksu et al., 2016; Bono et al., 2010; Cook et al., 2009; Grunwald and Bono, 2011; Grunwald et al., 2013; Matsuura and Stewart, 2004; Okada et al., 2009).”

4) Likewise, the authors give the impression as if they had contradicted the concept of importins functioning as chaperones for highly basic cargoes (Discussion section). The concept (Jäkel et al., 2002) implied that importins shield histones and ribosomal proteins against aggregation with polyanions and gave an intuitive explanation as to why import signals are not always as short as an SV40 NLS. It is unclear why the authors phrase their data as a contradiction. It would be more appropriate to give proper credit; the importin-chaperone concept should already be introduced in the Introduction.

We did not intend to contradict the concept of Importins functioning as chaperones for highly basic cargos. Instead, we want to expand the concept of chaperoning beyond the obvious charged-charged interactions since we observe many hydrogen bonds and hydrophobic interactions (many more than electrostatic contacts) in our Imp9•H2A-H2B complex. We revised the Discussion section to emphasize that we are not contradicting but supporting the previously reported Importin-chaperone concept by Jakel et al.

That paragraph now reads:

“Görlich and colleagues proposed in 2002 that negatively charged importins act as chaperones toward positively charged cargo proteins like histones (Jakel et al., 2002a). […] Kapβ2-FUS interactions are anchored through high affinity binding at the 26-residue PY-NLS linear motif of FUS that then enable weak, distributed and dynamic interactions with multiple mostly intrinsically disordered regions of FUS, to block formation of higher-order FUS assemblies and liquid-liquid phase separation (Yoshizawa, 2018).”

5) The referencing in the introduction is inappropriately biased. For example, there is no mentioning of the first discovered import pathway for a histone (Jakel, 1999). Likewise, the by now established fact that the RanGTPase system determines the direction of transport is referenced by two reviews of the Chook lab – despite the fact that the RanGTPase gradient concept had been worked out by a set of papers by the Görlich lab (see e.g. Izaurralde el al., 1997). Please amend this and similar problems.

We added the two references suggested by the reviewer to the revised Introduction.

6) I have little faith in the model of the RanGTP/ importin-9 complex. The Chook lab published a similar prediction before, namely a model of how RanGTP binds to the CRM1/ snurportin 1 complex (Dong et al., 2009). The experimental structure of the RanGTP/CRM1/ snurportin complex (Monecke et al., 2009), however, revealed that the model was not correct. I cannot see why a prediction of the RanGTP/ importin-9 prediction would be any better. There is less than 20% sequence identity to the most similar importin/ exportin with a solved RanGTP structure. Furthermore, the mode of RanGTP-binding is only partially conserved amongst the members of the importin β superfamily. Some conservation is evident in the contact regions of HEAT repeats 1-3. The additional contacts, however, are not conserved; they differ widely between the various solved RanGTP complexes, and I cannot see how one could possibly predict new ones in any reliable manner. A validation of the modeled interaction by mutagenesis will also not help because such validation will not assess the most problematic aspects of the model, namely contacts that have not been predicted. The RanGTP/ importin-9 structure model should be omitted because there is no reason to believe that it is reliable.

As the reviewer indicated, the N-terminal repeats (HEATs 1-4) of karyopherins, especially amongst importins, are conserved (we show this in a sequence alignment in Figure 4—figure supplement 4G). HEAT repeats 1-4 of the 7 importins shown in Figure 4—figure supplement 3A-F are of much more conserved (mean sequence identity of 20%) than the full-length importins (mean sequence identity of 17%). The locations of RanGTP binding, with its switch 1, switch 2 and helix 3 contacting importin HEATs 1-4, are clearly similar for Impβ, Kap95, Kapβ2, Kap121, Transportin-SR2 and Importin-13 (Figure 4—figure supplement 3A-F). Ran also binds exportins at equivalent sites on their HEATs 1-4 (data not shown). Examination of details of the Ran binding sites in Figure 4—figure supplement 4A-E and the sequence alignment Figure 4—figure supplement 4G show structural conservation of Ran binding at the N-termini of the importins.

In Figure 4—figure supplement 3G we show only the N-terminal HEAT repeats of Imp9, with H2A-H2B bound and Kap121•Ran aligned to mark the predicted Ran site, which looks just like the equivalent sites in panels A-F. This clearly marked “model” is shown to suggest that the Ran site on Imp9•H2A-H2B is most likely accessible. We do not show the bottom half of the Imp9•H2A-H2B structure so as not to imply how that half of Imp9 might bind RanGTP; we do not know the extent of how the superhelix of Imp9 might rearrange upon binding RanGTP. In Figure 4—figure supplement 3A-F, we observe that the superhelices/solenoids of the Importins adopt different pitches thereby placing different parts of the C-terminal halves of these Importins to interact with the α-4 helix and basic patch of RanGTP.

(Note: RanGTP was modeled onto the N-terminal HEAT repeats of the CRM1•SNUPN complex (Dong et al. NSMB 2009). The location of the predicted site at HEATs 1-4 of CRM1 resembles that in the Ficner/Görlich structure but the overall structure of our RanGTP•CRM1•SNUPN model looks different because of the large conformational change in the CRM1 superhelix that placed its C-terminal HEAT repeats to clamp RanGTP on the opposite side).

7) The signal to noise ratio in the ITC experiments is rather poor – resulting in rather broad confidence intervals of the fits. Some plots e.g. Figure 1—figure supplement 1D, E, G, H failed to reach saturation. Please supply a substantially improved dataset. Since the impact of ionic interactions and the magnitude of the effects are important for the argument, a dataset at physiological salt should be included. For measuring weak interactions, it will help to raise the concentration of the titrant to 30-50-fold higher than the analyte.

We took the reviewers’ concerns very seriously and repeated all ITC experiments using single batches of the different proteins to decrease variations that may arise from different preparations of protein reagents. The new ITC experiments were performed more rigorously with much higher quality proteins (gels, gel filtration traces and mass spectrometry data shown in this response document) in buffers with 150 mM NaCl. Every set of interactions was evaluated in triplicate and the data are consistently cleaner. The new data are summarized in the new Table 1 and the thermograms, traces and fits for the triplicate experiments shown in Figure 1—figure supplement 1.

Some interactions just do not result in high ΔH. Fortunately, none of our interactions are weak enough to warrant drastically raising titrant concentrations so we could avoid problems like precipitation and poor c-values that come from very high protein concentrations. Bad heats of dilution for some samples are something that plagues the entire histone field, and we have done what we can to address it.

8) Please identify the used H2A and H2B variants by including the relevant UniProt numbers.

After further consideration, we feel that the section on histones variants really does not fit in the current story. We removed this entire Results section. We will pursue more rigorous biochemical and cellular studies of Imp9 interactions and nuclear import of H2A and H2B variants for a future report.

9) Figure 1A (upper panel). Please identify the protein bands.

We re-did the experiment in Figure 1A and labeled the protein bands in new Figure 1A.

10) Figure 1B, please improve clarity of the cartoon.

We moved Figure 1B to the supplement (reviewer #1’s suggestion) and improved clarity of the cartoon by making it larger.It is now the new Figure 1—figure supplement 1A.

11) Figure 1E-G are too crowded to get the information. Please re-label to improve clarity, and please label only what is really needed for argument.

To improve clarity of the old Figures 1E-G (now new Figures 2B-D), we

1) Enlarged each figure panel.

2) Removed labels for many side chains that are partially hidden, thereby relieving the crowding.

We also added stereo figures of broader views for each of the three interfaces in the supplement. These new figures are now in Figure 2—figure supplement 1A-C.

We also added images showing omit map electron density of loops (omitted to calculate the omit maps) in each of the three interfaces. These new figures are now in Figure 2—figure supplement 2A-D.

12) Please clarify which form of Ran (wt vs. Q69L, yeast of human) is used for which experiment.

The information for the Ran used was already detailed in the Methods section of the original manuscript. We used S. cerevisiae Ran (Gsp1) residues 1-176 with the Q71L mutation. The truncated protein is stabilized in the GTP state and the Q71L mutation abolishes GTPase activity further stabilizing the GTP state. We added this information to the main text (Results section) and to the Figure 4 legend.

13) Fitting of the models into SAXS data is not very conclusive. How well do alternate coordinate models fit the SAXS data?

We removed the SAXS model from the new Figure 4. It is unclear how we should make alternate models for the Ran complex. But, independent of the model, the MW weights of the complexes determined by SAXS indicate that RanGTP is indeed bound to Imp9•H2A-H2B – we kept this data (upper portion of old Figure 3E) as the new Figure 4F.

14) The term "NLS" is ambiguous and by many (if not most) cell biologists considered to be an importin α-dependent nuclear import signal. I would therefore refrain from calling the importin 9-interacting interface an NLS. The more generic term "import signal" should cause less confusion.

We replaced “NLS” in the first paragraph of Discussion section with “extended linear nuclear import/localization motifs”.

Reviewer #3:

[…] Imp9 does not bind the NLS like sequences on the histone tail, but covers its surface. This deserves a a bit more discussion and a more rigorous analysis, including in ITC assays, as will be suggested below.

We added materials in the text on Imp9 covering the surface of the histone core rather than binding NLS-like tail sequences, in the following manner:

1) We expanded the description of the structures of the Imp9•H2A-H2B interfaces in Results section. We show and describe the weak electron density and very high B-factors of the small portion (5-residue segment) of the H2B tail that contacts Imp9 in Interface 2. We gave more details about Interfaces 1 and 3, including a new figure supplement (Figure 2—figure supplement 1D,) that shows many contacts made by the main chain of Imp9 to the histones.

2) We also show very consistent new mutagenesis/ITC data (Table 1 and Figure 1—figure supplement 1), which clearly show that the N-terminal tails of H2A and H2B tails do not contribute significantly to Imp9 binding. These data are described in the Results section.

3) We expanded discussion in the first and third paragraphs of Discussion section.

Additional experiments to test the existence of the Imp9-H2A-H2B complex in both cytoplasm and nucleus would strengthen the reported interaction at the level of IP in HeLa cell extracts.

Essential revisions:

ITC data:

- The results appear to have been derived from single measurements, see Figure 1—figure supplement 1A. As can be seen from this example, there are anomalies in data points in the important region of the titration where the heat of injection changes most. This indicates that the data point for full-length H2A-H2B cannot be trusted. Figure 1—figure supplement 1D, E, and F suffer from similar problems. I would urge the authors to conduct these assays in triplicate. In the absence of these data, the comparison between the distinct H2A-H2B constructs appears premature. I encourage the authors also to provide a stoichiometry value, as well as the ΔH and ΔS parameters from the fitting of the results.

We took the reviewers’ concerns very seriously and repeated all ITC experiments using single batches of each of the different proteins to decrease variations that may arise from different preparations of protein reagents. The new ITC experiments were performed much more rigorously with much higher quality proteins (gels, gel filtration traces and mass spectrometry data shown in this response document) in buffers with 150 mM NaCl. Every set of interactions was evaluated in triplicate experiments and the new data are consistently cleaner. The new data are summarized in the new Table 1 and the thermograms, traces and fits for the triplicate experiments are shown in Figure 1 —figure supplement 1.

In the SEDPHAT analysis, we fixed the stoichiomnetry in a 1:1 model. Therefore, instead of reporting stoichiometry, we report the Imp9 concentration-correction errors. We have provided ΔG, ΔS, and ΔH values in the new Table 1.

- The ΔH18-19 loop deletion construct shows a quite distinct ITC profile, the fitting here was carried out using an endothermic reaction, rather than fitting exotherms. This is not discussed in the manuscript, but ought to briefly be explained.

We made a brief mention of the endothermic vs. exothermic reaction of the ΔH18-19 loop deletion construct in Results section.

- Point mutants might also be quite instructive compared to the rather less surgical deletion of entire histone tails and loops, even if their impact to the overall binding energy may be limited.

Because of the extensive (and three separate) binding interfaces and the presence of many interactions in all three interfaces that involve main chain atoms, we agree with the reviewer that the impact of point mutants to overall binding energy will be limited. Because of the questionable utility of making Imp9 point mutants, we focused our energy on re-doing all the ITC analysis of deletion mutants of Imp9 and histones in the most rigorous manner.

(Note: considering the aforementioned limitations on the accuracy of the ITC experiments, providing four significant figures appears unjustified)

We decreased the significant figures of ITC parameters in the new Table 1.

Pulldown experiment between importin 9 and H2A-H2B constructs:

- Figure 2D lacks an appropriate negative control, such as an unrelated protein, MBP alone and/or an important mutant/distinct isoform that does not bind H2A-H2B.

Controls were added to the new Figure 4—figure supplement 1. We added a gel (Figure 4—figure supplement 1C) that shows controls of Imp9, H2A-H2B and RanGTP added to MBP immobilized on amylose resin. None of these proteins bind non-specifically to MBP or to the amylose resin. This includes H2A-H2B, which does not bind to immobilized MBP (lanes 6-10, Figure 4—figure supplement 1C).

For the gels in Figures 4A and 4B, we show the inputs and also the flow-through (FT) materials of all the pull-down experiments in Figure 4—figure supplement 1A, B. Both the inputs and FTs show the large excess and also the increasing concentrations of RanGTP used.

-The authors claim that the Imp9-H2A-H2B complex is present in the cytoplasm and nucleus and possibly also on chromatin. However, they do not directly show this. Co-IPs in these different cellular fractions would confirm this and would be a nice addition to the paper.

Co-IP experiments in the old Figure 1A (using whole cell lysates) were repeated with cytoplasmic and nuclear fractions. The new data is shown in the new Figure 1A. Imp9 is detected almost exclusively in the cytoplasm fraction where it co-IPs with H2BmCherry. Only a trace of Imp9 is detected in the nuclear fraction and none is detected in co-IP with nuclear H2BmCherry. The co-IP results are consistent fluorescence microscopy imaging in Figure 1B showing that Imp9 is mostly localized to the cytoplasm.

- Additional assays/evidence, such as imaging, showing that RanGTP can bind with Imp9-H2A/H2B in vivo would strengthen the manuscript.

We performed co-IP with H2BmCherry of the nuclear fraction. Ran is abundant in the nucleus and co-IPs with H2BmCherry. However, Imp9 is hardly detectable in the nuclear fraction even though it is abundant in the cytoplasmic fraction. Imp9 co-IP with H2BmCherry is detected only in the cytoplasmic fraction. Similarly, imaging also shows that Imp9 is found mostly in the cytoplasm. Imp9 is likely in the nucleus only transiently.

Gel shift assay in Figure 3B:

- As it stands, the current assay is difficult to interpret, as samples are not shown comparable on the same gel. In addition, the conditions that contain Imp9 are different, one is a preformed complex and the other is not. This also makes it difficult to compare activity and make firm conclusions based on this preliminary experiment. This can be readily experimentally addressed.

We performed new and extensive gel shift assays, showing all samples from an experiment on the same gel. These new results are shown in:

1) Figure 3D and E – Imp9 and DNA competing for H2A-H2B (ethidium bromide and Coomassie gels, respectively).

2) Figure 4D – Imp9 titrated to H2A-H2B to show formation of the Imp9•H2A-H2B complex and Ran to Imp9 or Imp9•H2A-H2B to show Imp9-Ran interactions and formation of the heterotetrameric Ran•Imp9•H2A-H2B complex.

3) Figure 5A and 5B – DNA is titrated to Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B, showing that DNA cannot compete for H2A-H2B from Imp9•H2A-H2B but can compete for H2A-H2B from RanGTP•Imp9•H2A-H2B to result in Imp9•RanGTP. Coomassie staining in 5A, and ethidium bromide staining in 5B.

4) Figure 5C and 5D – Imp9 or Imp9•Ran is titrated to DNA•H2A-H2B. Imp9 releases much more DNA than Imp9•Ran. Ethidium bromide staining in 5C, Coomassie staining in 5D.

5) Figure 5E – DNA/ethidium bromide gel of nucleosome assembly assays (protein gel in Figure 5—figure supplement 1B; controls in Figure 5—figure supplement 1A).

6) Figure 5F – DNA/ethidium bromide of nucleosome disassembly assays (protein gel in Figure 5—figure supplement 1C; controls in Figure 5—figure supplement 1A).

Figure 4—figure supplement 1:

- There seems to be less H2A/H2B when RanGTP is added. This experiment should be shown on one gel to compare conditions properly. In addition, this referee would suggest adding more RanGTP to test whether H2A-H2B levels decrease further.

We repeated the experiment by titrating increasing RanGTP concentrations, starting with 5 molar equivalents of RanGTP to MBP-Imp9•H2A-H2B in lane 6 of Figure 4A followed by 10 molar equivalents in lane 7, 20 molar equivalents in lane 8 and 30 molar equivalents in lane 9. Each of the four lanes show similar amounts of H2A-H2B and RanGTP bound to immobilized Imp9. Little H2A-H2B is displaced from the beads in the FT as shown in lanes 24-27 of the gel shown in Figure 4—figure supplement 1A.

The same amount of excess RanGTP was used in the experiment of MBP-PY-NLS binding to GST-Kapβ2 (Figure 4—figure supplement 1B). In this case, even 5 molar equivalents of RanGTP easily released all previously bound MBP-PY-NLS from the immobilized GST-Kapβ2.

[Editors' note: the author responses to the re-review follow.]

[…] We judge that there is not yet sufficient evidence that the RanGTP:Imp9:H2A:H2B complex is stable in solution, raising the question whether the proposed mechanism is correct that RanGTP binding to the Imp9:H2A:H2B complex is required for priming of the import complex that it can serve as a H2A:H2B donor during nucleosome assembly. Therefore, my criticisms of the revised manuscript refer primarily to the insufficient quality of newly included size-exclusion chromatography (SEC) interaction analysis that was introduced to support the formation of a stable RanGTP:Imp9:H2A:H2B tetramer.

Figure 4—figure supplement 1D. We had requested that the authors carry out additional SEC interaction experiments, ideally combined with a multi-angle light scattering analysis, that would clearly establish that RanGTP binding indeed results in the formation of a heterotetrameric RanGTP:Imp9:H2A:H2B complex, rather than triggering the release of the bound H2A:H2B import cargo and the formation of a heterodimeric Imp9:RanGTP complex, as it has been observed in countless other canonical import complex cases. This analysis is at the heart of the novelty of the presented manuscript. While the authors have included such a SEC analysis, the results of the experiment are uninterpretable. Either the quality of the experiment is insufficient, or the experiment is contrary to the author's conclusion.

The two SDS-PAGE gels of the preincubation run fractions of a mixture of Imp9:H2A:H2B with a 4-fold molar excess of RanGTP do not show stochiometric incorporation of RanGTP into the Imp9:H2A:H2B complex. As judged by the gel filtration profile of RanGTP in isolation, RanGTP elutes as two peaks, consistent with its known weak dimerization behavior: a major peak at 18.2 ml and a second much smaller peak at ~14 ml. The second smaller peak is close to the position where the Imp9:H2A:H2B heterotrimer elutes (12.9 ml). While there is a faint RanGTP band in the SDS-PAGE gel of the preincubation run (lanes 13-16), the band is clearly much weaker than the H2A:H2B bands, indicating at best a sub-stochiometric incorporation of RanGTP into the Imp9:H2A:H2B complex. However, it seems that the faint RanGTP band may not be the result of the incorporation of RanGTP into the Imp9:H2A:H2B complex, but is rather the secondary peak of RanGTP co-eluting independently. An SDS-PAGE gel of the RanGTP control injection is essential to distinguish these two possibilities. Moreover, there is a shift of ~0.4 ml in the elution profiles of RanGTP in isolation compared to the preincubation run profile (this is best seen in the blue and brown major peaks of RanGTP at an elution volume of ~18 ml). Such differences are common when experiments are carried out on different gel filtration columns, with different tubing lengths, or on different FPLC instruments. Additionally, the amounts of the injected proteins are unequal in the various injections and the baselines indicate that the column was not properly equilibrated between different injections. To definitively conclude whether RanGTP stoichiometrically incorporates into the Imp9:H2A:H2B complex, a proper SEC analysis needs to be carried out in which the various components are injected subsequently on the same gel filtration setup. High quality SDS-PAGE gels for all runs are essential for the interpretation of the experiment and should be included in the revised version of the manuscript.

At the request of the reviewer, we have performed new size exclusion chromatography (SEC) experiments to examine the interactions between Imp9•H2A-H2B and RanGTP. The SEC panel previously shown in Figure 4—figure supplement 1D of the old submission was an exploratory analysis performed very early in the project (in 2015), probably with somewhat subpar proteins. Our proteins preparations have improved tremendously since then. Here, we show two series of new SEC experiments in the new Figure 4—figure supplement 2.

The first SEC series include experiments (Figure 4—figure supplement 2A) with: (1) Individual proteins – 20 µM Imp9 alone and 60 µM RanGTP alone, (2) Imp9 + H2A-H2B 1:1 (20 µM), and (3) titrations of Imp9 + H2A-H2B 1:1 (20 µM) with Ran at 0.5 (10 µM), 1 (20 µM), 2 (40 µM) or 3 (60 µM) molar equivalents. Imp9 alone elutes at 13.6 mL, while the 1:1 Imp9•H2A-H2B complex elutes at 13.5 mL. When RanGTP is added, we see the formation of a 1:1:1 RanGTP•Imp9•H2A-H2B complex. Addition of an equimolar amount of RanGTP causes the Imp9•H2A-H2B peak to shift from 13.5 mL to 13.4 mL. Continued addition of RanGTP beyond a 1:1:1 mixture, results in the appearance of free RanGTP that elutes at 17.1 mL. Comparison to a 60 µM Ran alone control shows that the Imp9•H2A-H2B•RanGTP complex has a 1:1:1 stoichiometry. Quantitatively, the free RanGTP peak is absent in the 1:1:1 sample, is one-third of the control in a 1:1:2 sample, and two-thirds of the control in a 1:1:3 sample. The Ran only control shows that RanGTP is monomeric with no visible dimers. Free Ran elutes far from where Imp9 elutes.

The second SEC series is with proteins at higher concentration for SDS-PAGE visualization of eluted fractions (Figure 4—figure supplement 2B). We ran 1:1 Imp9 + H2A-H2B (70 µM) and 1:1:1 Imp9 + H2A-H2B + Ran (70 µM) on the same column with the same buffer as above, in two separate experiments. SEC of 1:1 Imp9 + H2A-H2B produces a single Imp9•H2A-H2B peak at elution volume of 13.5 mL. SEC of 1:1:1 Imp9 + H2A-H2B + Ran produces a single 1:1:1 RanGTP•Imp9•H2A-H2B peak eluting at 13.4 mL and SDS-PAGE of peak fractions shows the presence of Imp9, H2A-H2B, and Ran. Each protein stains in proportion to that seen in the input lane (note that the same molar equivalent of RanGTP in the input stains weaker than the histones), consistent with the formation of a 1:1:1 complex. Also, in the SEC there is no free H2AH2B or free Ran.

In conclusion, in the presence of Imp9, H2A-H2B and RanGTP, we see no free or released H2AH2B and we clearly see the formation of a 1:1:1 RanGTP•Imp9•H2A-H2B complex.

We also performed SEC analysis of the N-terminally truncated Imp9 mutant that was designed to not bind RanGTP, which we now use to replace the old pull-down binding assay that tested Ranbinding. Figure 4—figure supplement 6 panels C-E show SEC of the MBP-Imp9D1-144 mutant: C) + RanGTP, D) + H2A-H2B, and E) + H2A-H2B and RanGTP. SEC analysis shows that RanGTP does not interact with the Imp9 mutant. No interaction is seen at μM concentrations even when RanGTP is added at a 6-fold molar excess. This is obvious from the SDS-PAGE analysis of SEC fractions, showing that the Imp9 mutant and RanGTP do not comigrate (Figure 4—figure supplement 6C). The Imp9 mutant protein is functional as the interaction is maintained with H2A-H2B. This is consistent with the crystal structure showing that the region spanning HEAT repeats 1-3 of Imp9 (residues 1-144) is only a very small portion of the very large Imp9•H2A-H2B interface (Figure 4—figure supplement 6D). Not surprisingly, like Imp9 mutant alone in Figure 4—figure supplement 6C, the histone-bound Imp9 mutant also does not bind RanGTP when the GTPase is added at a molar excess (Figure 4—figure supplement 6E).

SEC details are described in the Materials and methods section.

Unfortunately, the poor quality of the newly included SEC analysis casts further doubts on the validity of the SAXS analysis of the RanGTP:Imp9:H2A:H2B complex. At a minimum the current SEC analysis seems to indicate that the RanGTP:Imp9:H2A:H2B heterotetramer is non-stochiometric at the tested Imp9:H2A:H2B concentration of 100 μM. The maximum reported concentration of the RanGTP:Imp9:H2A:H2B complex in the SAXS analysis was 30 μM. Because no stochiometric RanGTP:Imp9:H2A:H2B tetramer was observed in SEC analysis at an injected concentration of 100 μM, which is comparable to the SAXS analysis, if one considers that injected samples are typically ~4-fold diluted on a gel filtration column, it seems unlikely that the SAXS analysis was carried out with a monodisperse sample of the RanGTP:Imp9:H2A:H2B heterotetramer. Unfortunately, the methods provide no information how the authors reconstituted the RanGTP:Imp9:H2A:H2B tetramer for their SAXS analysis. Generally, monodispersity and the formation of a stable species is essential for a meaningful SAXS analysis. Therefore, SAXS experiments are typically carried out either directly following elution from a gel filtration column or better yet by directly coupling the gel filtration column to the SAXS cell.

Our new SEC analysis, performed with 20 µM or 70 µM protein concentrations in the input, show stable 1:1:1 RanGTP•Imp9•H2A-H2B complex. Per the reviewer’s assumption of ~1 in 4 dilution in the eluate peak of an SEC experiment, the 1:1:1 RanGTP•Imp9•H2A-H2B complex appears to be stable even down at 5 µM (Figure 4—figure supplement 2A). Our SAXS samples of Imp9, Imp9•H2A-H2B, Imp9•RanGTP, and RanGTP•Imp9•H2A-H2B varied in concentrations as high as 31-43 µM, which are all significantly more concentrated than in our SEC analysis.

Details of SAXS sample preparation:

We include a new paragraph in Materials and methods section with details of SAXS sample preparation. All SAXS samples were prepared by SEC to exchange the proteins into SAXS buffer and to remove excess proteins. We show the profiles of SEC runs (performed in July 2017) that produced the SAXS samples in Figure 1. SDS-PAGE/Coomassie-staining of the fractions were performed to ensure proper formation of complexes but the gels were not recorded or kept so regrettably cannot be shown. SEC to prepare the SAXS samples were also performed on a Superdex 200 column but not the same column used in the new Figure 4—figure supplement 2. The buffers are also different, as SEC preparations for SAXS were performed in SAXS buffer (20 mM HEPES pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, and 10% glycerol). Nevertheless, the trends of elution volumes are similar with the elution volumes of RanGTP•Imp9•H2A-H2B > Imp9 binary complex > Imp9 (see Figure 1A) with no peaks for free histones or free RanGTP in the second SEC for the preformed RanGTP•Imp9•H2A-H2B sample (Figure 1B).

After the SEC analysis, all our SAXS samples were further improved through multiple steps, right before the data collection at the SSRL SAXS beamline, as follows:

Author response image 1
SEC for SAXS sample prepation.

A. SEC profiles of 1) Imp9 alone (blue trace), 2) a previously purified Imp9•RanGTP complex (light blue trace) and 3) a previously purified Imp9•H2A-H2B + excess RanGTP (green; the peak at ~ 16 ml is excess RanGTP). Elution volume for each of the Imp9-containing peaks is listed. B. Fractions for the Imp9 containing peak in the SEC of Imp9•H2A-H2B + excess RanGTP (green trace in A) were pooled and subjected to a second round of SEC to produce the SAXS sample for the heterotetrameric complex.

https://doi.org/10.7554/eLife.43630.025

First, the 10% glycerol in the SAXS buffer protects the protein samples from any potential radiation damage during X-ray exposure (Kuwamoto et al., 2004) (also see Figure 4—source data 1). Our earlier studies show that 5-20% (v/v) glycerol concentrations do not affect protein compaction (Yoshizawa et al., 2018).

Second, all protein and buffer solutions were filtered through 0.1 µm membranes (Millipore) to remove any aggregates, right before each measurement at the SAXS beamline (LoPiccolo et al., 2015).

Third, we collected SAXS profiles at multiple concentrations ranging from 0.5 to 5.0 mg/ml, then the final merged SAXS profiles were basically obtained by extrapolation to zero concentration, eliminating any concentration dependence affected by potential aggregation.

We achieved a high level of monodispersity, with no aggregation or degradation/dissociation, for our SAXS samples. This high level of monodispersity is independently determined from the SAXS analysis. Linear Guinier plots (rightmost panels, Figure 4—figure supplement 3A-D) for all 4 samples and consistency of molecular weight estimations (Figure 4G and Figure 4—source data 1, Figure 4—source data 2) report directly and independently on the monodispersity and the stability of the SAXS samples. In other words, SAXS analysis provides an internal readout on the monodispersity of the samples that are being analyzed, independent of data from sample preparation by SEC. SAXS is by far the more rigorous biophysical analysis and a quantitative one compared to SEC.

We explain further the relevant SAXS analysis (Guinier plots and MM determination) in detail here:

1) The linearity of a Guinier plot reports on monodispersity of the sample, in terms of the particle size as supported by basic SAXS theories. If there was a mixture of species of particles (e.g. any mixture of Ran•Imp9•histones, Imp9•histones, Imp9•Ran, Imp9 and Ran) in solution, a final SAXS profile would become a population-weighted combination from each component in the mixture. In other words, each component in the mixture would contribute to the Guinier plot with a different slope value (corresponding to its Rg value), leading to a curvature or non-linearity in the Guinier plot. Consequently, the Guinier plot cannot be and is not linear when the sample contains a mixture of particles with different sizes. We did not observe such a curvature or non-linear pattern in any of the Guinier plots obtained from our SAXS data (rightmost panels, Figure 4 —figure supplement 3A-D). Our Guinier plots look very good and linear, indicating that all our SAXS samples were highly monodisperse, homogeneous and stable in solution.

2) The molecular weight estimated (MM or MW(SAXS) in Figure 4G) from the SAXS profiles of the RanGTP•Imp9•H2A-H2B sample is consistent with having 1 copy of each of the 4 polypeptide chains in RanGTP•Imp9•H2A-H2B, which further supports formation and stability of that complex. Our calculation of MM from the extrapolated scattering intensity at zero angle I(0) using SAXSMOW should have an uncertainty of <10% (Piiadov et al., (2018) and Fischer et al., (2010).

3) According to an article by the Svergun group, a worldwide leading group in SAXS, "one of the most important overall parameters, which can be derived from small-angle X-ray scattering (SAXS) experiments on macromolecular solutions is the molecular mass (MM) of the solute. In particular, for a monodisperse protein solution, MM of the solute is calculated from the extrapolated scattering intensity at zero angle I(0). Assessing MM by SAXS provides valuable information about the oligomeric state and absence of unspecific aggregation in solution. The value of MM can either be estimated by comparison with a protein standard with a known MM or by determining the absolute scattering intensity using, e.g., water scattering. In both cases, knowledge about the solute concentration and about the partial specific volume of the protein is required. […] One of the most common applications of SAXS is the determination of the oligomeric state of the biomolecule (e.g. a protein or a macromolecular complex) or monitoring of aggregation or degradation processes, which can be readily done by assessing the MM value." (Mylonas and Svergun, (2007)).

The observed difference of the major sedimentation coefficient peaks in the AUC analysis of the Imp9:H2A:H2B complex in the absence and presence of a 3-fold molar excess of RanGTP seems to indicate heterotetramer formation. However, AUC analyses are typically performed at much lower concentrations than gel filtration interaction analyses (the authors also provide here no detail in the methods about their employed concentrations). While the pull-down and AUC data seem to support the interpretation that RanGTP is indeed incorporated into the Imp9:H2A:H2B complex without releasing the H2A:H2B cargo, I am puzzled why the gel filtration data does not support this conclusion.

Individual purified proteins Imp9, H2A-H2B and RanGTP were dialyzed into AUC buffer before mixing them for AUC. Samples for AUC contain: 1) 450 µL Imp9 alone (3 µM), 2) 450 µL RanGTP alone (10 µM), 3) 450 µL H2A-H2B (10 µM), 4) 3 µM Imp9 + 3 µM RanGTP in a total volume of 450 µL, 5) 3 µM Imp9 + 3 µM H2A-H2B in a total volume of 450 µL, 6) 3 µM Imp9 + 3 µM H2AH2B+10 µM RanGTP in a total volume of 450 µL. The proteins were mixed overnight before loading into the AUC cell.

We have added these details of sample preparation to methods (pages 60-61). Proteins used for AUC are only slightly lower in concentration (3 µM) than from SEC analysis ((~5 µM). Both AUC and SEC analyses show RanGTP interacting with Imp9•H2A-H2B. AUC is by far the more rigorous biophysical analysis and a quantitative one compared to SEC. AUC shows the RanGTP•Imp9•H2A-H2B to be a larger assembly (4.6 S) than Imp9•H2A-H2B (4.3 S).

The recommendation is that the authors carry out a proper gel filtration interaction analysis as outlined above. As an important control, the SEC interaction analysis should also include the truncated Imp9:H2A:H2B complex that the authors identified to be deficient in RanGTP binding.

Figure 4—figure supplement 6 panels C-E show SEC of the MBP-Imp9D1-144 mutant in the presence of excess RanGTP, excess H2A-H2B, and of the purified MBP-Imp9D1-144•H2A-H2B + 3-fold molar excess RanGTP, all performed in buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT and 10% glycerol. The MBP-Imp9D1-144 mutant does not bind RanGTP but is functional as it binds H2A-H2B. Consistently, MBP-Imp9D1144•H2A-H2B also shows no interactions with RanGTP.

Additionally, the entire SAXS analysis should be removed from the manuscript and the structural characterization of the RanGTP:Imp9:H2A:H2B complex and the likely associated conformational change upon RanGTP binding should be published separately in a future study. Alternatively, an EM analysis could be included but I do not think this is necessary.

We have performed extensive SEC analysis with many controls and at different protein concentrations, all of which show that 1) RanGTP does not dissociate H2A-H2B from Imp9 and 2) RanGTP binds the Imp9•H2A-H2B complex to form a 1:1:1 RanGTP•Imp9•H2A-H2B complex even in at an estimated concentration of ~5 µM. Compared to SEC, AUC analysis is a much more quantitative and rigorous method that shows the 3 µM RanGTP•Imp9•H2A-H2B complex to be larger than the Imp9•H2A-H2B of the Imp9•RanGTP complexes. SAXS samples were prepared by SEC and are of much higher concentrations (31-43 µM) than either AUC samples or the ~5 µM 1:1:1 stable RanGTP•Imp9•H2A-H2B complex that eluted from SEC. Most importantly, the ability of SAXS to accurately determine molecular weight of the macromolecular particle in a buffer with complex composition (e.g. buffers with glycerol that are critical for solubility of karyopherins) is superior to AUC and most certainly superior to SEC (method is subjected to problems such as shape of particle, interactions with column, viscosity/density of solution and macromolecular dissociation from shear force). SAXS analysis independently assesses mono/poly-dispersity of protein samples. Our SAXS analysis show that our samples, including that of the RanGTP•Imp9•H2A-H2B one, are highly monodisperse and the RanGTP•Imp9•H2A-H2B complex is stable with a MW(SAXS) or MM determined from SAXS profiles that matches a 1:1:1 assembly of Ran, Imp9 and H2A-H2B. SAXS analysis is in fact the most important complement to our AUC, SEC, EMSA and pull-down binding assays, and should not be removed from the manuscript.

We are unsure what aspect of "structural characterization of the RanGTP•Imp9•H2A-H2B complex” the reviewer is referring to. We have not solved or presented any structures of the RanGTP•Imp9•H2A-H2B complex. The closest thing to a “structure” in the previous submission was an ab initio SAXS model in a supplement figure. We have removed all ab initio SAXS models in Figure 4—figure supplement 3A-D. In the previous version of the manuscript, we also placed a cartoon/ribbon diagram of RanGTP into the N-terminal region of Imp9 through structural alignment of HEAT repeats 1-4 of Kap121 with Imp9. We have now removed the cartoon of RanGTP and replaced it with a schematic diagram to show the approximate placement of Ran from alignment with the Kap121•RanGTP structure (new Figure 4—figure supplement 6A). In a complementary figure we show only the structure of Imp9•H2A-2B that we solved and colored Imp9 residues at the predicted Ran-binding site green (new Figure 4—figure supplement 6B).

It is clear from DNA competition and nucleosome assembly/disassembly assays that RanGTP binding to Imp9•H2A-H2B changes the interactions between Imp9 and H2A-H2B. We therefore make a reasonable speculation that "the likely associated conformational change upon RanGTP binding” changes interactions between Imp9 and H2A-H2B. This speculation is limited to a single sentence in Discussion that states “Accessibility of the N-terminal HEAT repeats of Imp9 in the histones complex may allow formation of the RanGTP•Imp9•H2A-H2B complex, but proximity of the Ran and histones binding sites coupled with the flexibility of the HEAT repeats architecture of Imp9 and the propensity for conformational changes likely changed the kinetics of Imp9-histone binding.” We did not change this sentence. It would be intellectually lazy of us to not discuss or speculate possible ways Ran binding could influence Imp9•H2A-H2B interactions.

https://doi.org/10.7554/eLife.43630.030

Article and author information

Author details

  1. Abhilash Padavannil

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Investigation, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
  2. Prithwijit Sarkar

    Department of Biological Sciences, University of Texas at Dallas, Richardson, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  3. Seung Joong Kim

    Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Tolga Cagatay

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Jenny Jiou

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Chad A Brautigam

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  7. Diana R Tomchick

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Validation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7529-4643
  8. Andrej Sali

    1. Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, United States
    2. Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, United states
    Contribution
    Supervision, Funding acquisition, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0435-6197
  9. Sheena D'Arcy

    Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, United States
    Contribution
    Formal analysis, Supervision, Funding acquisition, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5055-988X
  10. Yuh Min Chook

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    yuhmin.chook@utsouthwestern.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4974-0726

Funding

National Institute of General Medical Sciences (U01GM98256-01)

  • Andrej Sali
  • Yuh Min Chook

National Institute of General Medical Sciences (R01GM083960)

  • Andrej Sali

National Institute of General Medical Sciences (P41GM109824)

  • Andrej Sali

National Institute of General Medical Sciences (R01GM112108)

  • Andrej Sali

University of Texas at Dallas (Start-up funds)

  • Sheena D'Arcy

National Institute of General Medical Sciences (R01GM069909)

  • Yuh Min Chook

Welch Foundation (I-1532)

  • Yuh Min Chook

Leukemia and Lymphoma Society (Scholar Award)

  • Yuh Min Chook

University of Texas Southwestern Medical Center (Endowed Scholars Program)

  • Yuh Min Chook

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Bing Li for plasmids expressing H2A and H2B, Hongtao Yu for the H2BmCherry stable cell lines, Binita Shakya for Ran protein, James Chen for advice on X-ray structure determination, and the Structural Biology Laboratory and Macromolecular Biophysics Resource at UTSW for their assistance with crystallographic and biophysical data collection. Crystallographic results are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER) under contract DE-AC02-06CH11357. We thank T Matsui and TM Weiss at SSRL, SLAC National Accelerator Laboratory, for assistance with collecting SAXS data. SAXS experiments were performed at the SSRL, SLAC National Accelerator Laboratory operated for DOE by Stanford University. The SSRL SMBP is supported by the DOE BER, by the National Institutes of Health (NIH), NCRR, Biomedical Technology Program (P41RR001209), and by NIGMS, NIH (P41GM103393). This work was funded by NIGMS of NIH under Awards R01GM069909 (YMC), U01GM98256-01 (YMC and AS), R01GM083960 (AS), P41GM109824 (AS), R01GM112108 (AS), the Welch Foundation Grants I-1532 (YMC), the Leukemia and Lymphoma Society Scholar Award (YMC), start-up funds from the University of Texas at Dallas (SD) and the University of Texas Southwestern Endowed Scholars Program (YMC).

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Germany

Reviewing Editor

  1. Andrea Musacchio, Max Planck Institute of Molecular Physiology, Germany

Publication history

  1. Received: November 14, 2018
  2. Accepted: March 9, 2019
  3. Accepted Manuscript published: March 11, 2019 (version 1)
  4. Version of Record published: April 8, 2019 (version 2)

Copyright

© 2019, Padavannil et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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