1. Structural Biology and Molecular Biophysics
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Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology

  1. Xiao Tao
  2. Roderick MacKinnon  Is a corresponding author
  1. The Rockefeller University, Howard Hughes Medical Institute, United States
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Cite this article as: eLife 2019;8:e52164 doi: 10.7554/eLife.52164

Abstract

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

Introduction

Voltage sensors are integral membrane proteins that ‘sense’ transmembrane voltage differences. They exist as regulatory domains on voltage-gated ion channels and voltage-dependent phosphatase enzymes (Hille, 2001; Murata et al., 2005). All voltage sensors have a shared structure consisting of four helical transmembrane segments (Noda et al., 1984; Papazian et al., 1987; Tanabe et al., 1987; Tempel et al., 1987; Jiang et al., 2003a). The fourth helix, termed S4, contains positive-charged amino acids that move within the transmembrane electrical field as part of a global conformational change (Noda et al., 1984; Tanabe et al., 1987; Tempel et al., 1987; Aggarwal and MacKinnon, 1996; Seoh et al., 1996; Long et al., 2005a; Long et al., 2005b; Long et al., 2007; Swartz, 2008; Tao et al., 2010; Guo et al., 2016; Shen et al., 2017; Wisedchaisri et al., 2019; Xu et al., 2019). Sensor conformational changes are imparted onto the channel or enzyme to regulate activity.

Atomic structures of several different eukaryotic voltage-gated K+ (Kv) channels demonstrate that nature employs two topologically distinct connections between the voltage sensors and the ion channel pore (Long et al., 2005a; Whicher and MacKinnon, 2016; Sun and MacKinnon, 2017; Tao et al., 2017; Wang and MacKinnon, 2017). Viewed down an axis perpendicular to the membrane, Kv channels are four-fold symmetric, exhibiting one central pore surrounded by four voltage sensors. Each subunit encodes a voltage sensor from transmembrane helices S1-S4 and one fourth of the pore from S5 and S6. The channel tetramer is held together by a ‘barrel stave’ assemblage of the pore, with the voltage sensors residing on the periphery (Long et al., 2005a). The topological distinction between two classes of Kv channels relates to whether the voltage sensors are ‘domain-swapped’ or not. If four K+ channel subunits are designated A, B, C and D, to be domain-swapped means the voltage sensor of subunit A leans against the pore-forming helices of subunit B, B against C and so on around the tetramer, as shown in Figure 1A. In non-domain-swapped Kv channels, the voltage sensor of subunit A leans against the pore-forming helices of subunit A, B against B, etc., as shown in Figure 1B. Kv1-7 channels have domain-swapped voltage sensors; Kv10-12 and Slo1 channels have non-domain-swapped voltage sensors (Long et al., 2005a; Whicher and MacKinnon, 2016; Sun and MacKinnon, 2017; Tao et al., 2017; Wang and MacKinnon, 2017). Other voltage-gated channels can also be characterized according to this classification: voltage-gated Na+ (Nav) and Ca2+ (Cav) channels and other cation channels have domain-swapped voltage sensors (Payandeh et al., 2011; Zhang et al., 2012; Guo et al., 2016; Wu et al., 2016; Shen et al., 2017; Yan et al., 2017; She et al., 2018); HCN channels, which are hyperpolarization-activated cation channels, have non-domain-swapped voltage sensors (Lee and MacKinnon, 2017).

Two distinct topological connections between the voltage sensors and the ion channel pore exemplified by Kv1.2–2.1 paddle chimera (PDB: 2R9R) (A, C) and eag1 (Kv10) (PDB: 5K7L) (B, D) channel, viewed along the 4-fold axis from the extracellular side (A, B) or parallel to the membrane with the extracellular side up (C, D).

The channels are shown as ribbons and each subunit of a tetramer is colored uniquely. In panels C and D, voltage sensors of the front and back subunit are omitted for clarity. K+ ions are shown as cyan spheres.

The difference between domain-swapped and non-domain-swapped topologies is much more than an architectural curiosity. In the former case, the voltage sensor is connected to the pore by an ~20 Å long S4-S5 linker helix, which runs along the channel’s cytoplasmic surface and contacts the S6 helix, which forms the gate (Figure 1C) (Long et al., 2005a; Long et al., 2007). Conformational changes within the voltage sensor are transmitted to the gate through the S4-S5 linker helix (Long et al., 2005b; Guo et al., 2016; Wisedchaisri et al., 2019; Xu et al., 2019). Channels with non-domain-swapped voltage sensors do not have a helical S4-S5 linker and the mechanism of gate control is unknown (Figure 1D).

KvAP is a Kv channel from the thermophilic archaea Aeropyrum pernix (Ruta et al., 2003). It provided the first atomic structures from a voltage-gated ion channel and the isolated voltage sensor structure turned out to be the prototype for this domain family (Jiang et al., 2003a). Crystallographic analysis of the full-length KvAP channel, however, consistently yielded structures with voltage sensors that were rotated relative to the pore and partially unfolded (Jiang et al., 2003a; Lee et al., 2005). Together with voltage-dependent accessibility measurements, the KvAP structures led us to propose the ‘paddle model’, in which S4 moves adjacent to the lipid membrane as part of a helix-turn-helix structure consisting of helices S3b and S4 (Jiang et al., 2003b).

Until recently, all voltage-gated channels were thought to be of the domain-swapped variety (Whicher and MacKinnon, 2016). For this reason, KvAP was considered representative of the more extensively studied Shaker-like (i.e. Kv1) channels, with domain-swapped voltage sensors (Long et al., 2005a). We show here with a cryo-EM structure that this is not the case. In addition to being a non-domain-swapped Kv channel, KvAP has other structural features that distinguish it further from Shaker and most other voltage-gated ion channels. Structural differences between KvAP and Shaker may account for some discrepant results in the study of their voltage sensor conformational changes.

Results

Image analysis and map calculation

KvAP was expressed in E. coli, extracted in a mixture of lauryl maltose neopentyl glycol (LMNG) and cholesteryl hemisuccinate (CHS), exchanged into digitonin and then purified as a complex with Fab fragments using size exclusion chromatography (Figure 2—figure supplement 1). The Fab fragments, which bind to KvAP’s voltage sensors, were used to assist the alignment of channels in image processing. Following 2D classification, 734,850 particles were 3D-classified in Relion3 with C1 symmetry (Figure 2—figure supplement 2) (Scheres, 2012). All classes showed that the voltage sensors were oriented with Fabs projecting towards the extracellular face of the channel. This orientation is compatible with the extracellular accessibility of these Fabs in electrophysiological studies (Jiang et al., 2003b). The extracellular orientation of Fabs in cryo-EM images contrasts with the non-native orientation of KvAP voltage sensors in crystal structures of the full-length channel, in which detergents more dispersive than digitonin were used (Jiang et al., 2003a; Lee et al., 2005).

While the Fabs were on the extracellular surface in all 3D classes, the precise positioning of the Fabs was variable, as shown (Figure 2—figure supplement 2B). The positional variability explains why in both 2D (Figure 2—figure supplement 3A,B) and 3D classes (Figure 2—figure supplement 2B, Figure 2—figure supplement 3C), density for the four Fabs is not constant: in some classes only a single Fab is well aligned, in others 2, 3, or least frequently 4 Fabs are aligned. The variation in Fab orientation is consistent with past studies showing the high degree of mobility of the S3b-S4 paddle region of the voltage sensor (Ruta et al., 2005; Butterwick and MacKinnon, 2010). To increase resolution of the structure we selected one class, representing 13% of the particles, in which all four Fabs were oriented most similarly with respect to each other. We emphasize that these particles were not fundamentally different than the others, but statistically they happened to have more similarly-oriented Fabs, thus permitting better particle averaging. To analyze the pore and voltage sensors in greater detail, we further applied focused 3D classification in Relion3 while masking the KvAP transmembrane region and Fabs and also applying C4 symmetry (Figure 2—figure supplement 2B). The final density map, calculated to 6 Å resolution, showed continuous density for the transmembrane region, density for the Fabs on the extracellular side and a poorly resolved cytoplasmic domain (CTD) on the intracellular side (Figure 2, Figure 2—figure supplement 2, and Figure 2—figure supplement 4). The existence of the cytoplasmic domain, formed by an extension of the C-terminus, was predicted from protein synthesis studies of KvAP (Figure 2—figure supplement 4C) (McDonald et al., 2019).

Figure 2 with 5 supplements see all
Cryo-EM map of KvAP and model generation.

(A) The isolated KvAP voltage sensor crystal structure (PDB: 1ORS, shown as red ribbons) docked into the cryo-EM map (gray mesh), viewed in stereo, parallel to the membrane with the extracellular side up. (B) Same representation as panel (A), rotated 180° around the vertical axis. (C) The pore domain of a single subunit from the crystal structure of the full-length KvAP (PDB: 1ORQ, shown as red ribbons) docked into the cryo-EM map (gray mesh), viewed in stereo, parallel to the membrane with the extracellular side up. (D) The pore domain of the full-length KvAP crystal structure (PDB: 1ORQ, shown as ribbons with each subunit colored uniquely) docked into the cryo-EM map (gray mesh), viewed from the cytoplasmic side. (E) Overall structure of KvAP, viewed along the 4-fold axis, from the extracellular side. The channel is shown as ribbons and each subunit of the tetramer is colored uniquely. K+ ions are shown as cyan spheres.

Structure of KvAP

A model corresponding to the crystal structure of the isolated voltage sensor, unmodified, was placed into the density (Figure 2A,B) (Jiang et al., 2003a). We note that this model is similar to a second structure that we determined independently using NMR (Butterwick and MacKinnon, 2010). A model corresponding to the crystal structure of the pore, unmodified, was also placed into the density (Figure 2C,D) (Jiang et al., 2003a). Small rigid body adjustments of helical segments were made, followed by a round of real-space refinement. The only new model rebuilding at the level of individual amino acids involved four amino acids inside the continuous density connecting S4 to S5. Without the crystal structures it would have been impossible to build more than a poly-alanine model at 6 Å resolution. However, the crystal structures, which were determined at 1.9 Å for the voltage sensor and 3.2 Å for the pore, conform so well to the experimental map (Figure 2) that we know the approximate location of the amino acids in the final model.

An important first conclusion is that KvAP is a non-domain-swapped voltage-gated ion channel, meaning each voltage sensor is contiguous with the pore element from the same polypeptide chain (Figure 2E). That this must be the case is evident in the short turn connecting S4 to S5 instead of a long α-helical S4-S5 linker (Figure 2C). Therefore, KvAP is topologically like Kv10-12 channels and unlike Kv1-7 channels (Figures 1 and 2E).

Figure 3 highlights a notable structural difference between KvAP and eag1 (Kv10) even though they both share a non-domain-swapped topology. Viewed from the extracellular side, their voltage sensors orient differently with respect to the pore, as shown (Figure 3A). The unique orientations result from the manner in which the S1 from the voltage sensor and S5 from the pore interact with each other. In eag1, S1 contacts S5 near the extracellular surface, but is displaced about 15 Å away from S5 near the intracellular surface (Figure 3C). In contrast, S1 in KvAP runs parallel to and in contact with S5 throughout its length inside the membrane (Figure 3B). Perozo and co-workers analyzed amino acid side-chain accessibility and mobility in membrane-embedded KvAP channels using EPR (Cuello et al., 2004). Their data, which we have mapped onto the structure of KvAP, support the S1-S5 contact observed (Figure 4). Specifically, a continuous surface of low side-chain mobility and low O2 collision frequency, which are both signatures of a buried protein surface, is observed where S1 contacts S5 in the cryo-EM structure. Altogether, the cryo-EM structure is consistent with the EPR data. This consistency suggests that the cryo-EM structure is representative of a conformation of KvAP inside lipid bilayers.

Comparison of KvAP and eag1 structures.

(A) Overlay of KvAP (colored red) and eag1 (PDB: 5K7L, colored blue), viewed along the 4-fold axis, from the extracellular side. The two channels are aligned with respect to the pore domain (shown as Cα traces) and the VSDs are shown as surfaces. For eag1, only the transmembrane region is shown for clarity. (B) Ribbon representation of a single subunit of KvAP, viewed in stereo, parallel to the membrane with the extracellular side up. (C) Ribbon representation of a single subunit of eag1, viewed in stereo, parallel to the membrane with the extracellular side up. Only the transmembrane region is shown for clarity.

Cryo-EM structure of KvAP is consistent with the EPR data in lipid vesicles.

(A,B) Ribbon representation of a single subunit of KvAP, viewed parallel to the membrane with the extracellular side up. Panel B is rotated 180° from panel A around a vertical axis. (C–F) Remapping of EPR data digitized from Cuello et al. (2004) onto a single subunit of KvAP (pore domain as gray ribbons, voltage sensor domain as gray surface) in the same views as panels A and B (Cuello et al., 2004). Panels C and D show the O2 accessibility (red) and panels E and F show the mobility (green) from reconstituted full-length KvAP.

Conformational state

Figure 5 shows side views of the KvAP model, excluding the intracellular domain. In electrophysiological studies on KvAP, the membrane must be depolarized first before the extracellularly-applied Fabs can bind (Jiang et al., 2003b). In the structure, the helical turn of S3b into S4, the Fab epitope, is exposed to the extracellular surface, compatible with Fab accessibility and a depolarized conformation of KvAP. The voltage sensor is similar in conformation to depolarized voltage sensors in other voltage-gated ion channels (Figure 5—figure supplement 1). However, because of the unique interaction between S1 and S5, the S3b-S4 paddle in KvAP resides on the outer-most perimeter of the channel, oriented tangent to the pore domain.

Figure 5 with 2 supplements see all
Conformational state of KvAP.

(A) Overall structure of KvAP viewed parallel to the membrane with the extracellular side up. The channel is demarcated by a gray translucent surface and voltage sensors, S4-S5 linkers and intracellular part of S5 are shown as cyan ribbons. Blue spheres show Cα positions of the six positive-charged residues on S4: R117, R120, R123, R126, R133 and K136 from top to bottom. Yellow and two red spheres indicate Cα positions of the gating charge transfer center (yellow: Leu69; Red: Asp72 and Glu93). Gray bars approximate the location of membrane surfaces. (B) Stereo view of KvAP in the same orientation as in panel (A), with the pore domain shown as a gray surface and the voltage sensor domains, the S4-S5 linkers and the intracellular part of S5 as cyan ribbons. (C) Zoomed-in view of one voltage sensor in the same representation as panel (B). Side-chains of the six positive-charged residues as well as the gating charge transfer center are shown as sticks and colored according to atom type.

The pore is less open than in the crystal structure of the full-length channel, but more open than in other closed K+ channels (Figure 2D, Figure 5—figure supplement 2) (Jiang et al., 2003a; Tao et al., 2009). Functionally, KvAP opens and then inactivates partially following membrane depolarization (Schmidt et al., 2009). The conformation of the inner helix gate in this structure might reflect a mixture of open and inactivated channels.

Remapping of biotin-avidin accessibility data

Figure 6 maps previous data on the accessibility of tethered biotin to avidin onto the KvAP structure (Ruta et al., 2005; Banerjee and MacKinnon, 2008). Red and blue spheres show accessibility to the extracellular and intracellular sides of the membrane, respectively. Black spheres show positions inaccessible to either side. With a 1 Å length tether, only superficial sites near the aqueous surfaces are accessible, except at two positions, shown as blue spheres for intracellular accessibility, on the S4 helix. These outlier positions on the structure are more than 15 Å away from the intracellular surface. But the accessibility assay was carried out in membranes under voltage clamp. The result implies that the KvAP voltage sensor can undergo very large voltage-dependent conformational changes to bring these positions close to the intracellular surface. Experiments using longer tethers show results consistent with shorter tethers. Specifically, positions on S4 become exposed to both the extracellular and intracellular sides, depending on the membrane voltage (Figure 6—figure supplement 1).

Figure 6 with 1 supplement see all
Remapping of biotin-avidin accessibility data onto the KvAP cryo-EM structure.

Previous data on tethered biotin-avidin accessibility with 1 Å effective tether length is remapped onto the cryo-EM structure of KvAP (Ruta et al., 2005; Banerjee and MacKinnon, 2008). Only two opposing subunits (gray and orange ribbons) are shown for clarity. Red and blue spheres represent positions accessible to the extracellular and intracellular sides of the membrane, respectively. Black spheres represent positions inaccessible to either side of the membrane.

Discussion

The surprising new finding presented in this paper is that the voltage sensors in KvAP are not domain-swapped. We had assumed incorrectly that KvAP and Kv1 were similar in structure, but they are not (Lee et al., 2005; Ruta et al., 2005). Through studies of domain-swapped Kv, Nav, Cav and other voltage-gated ion channels we are starting to understand certain aspects of how they work (Long et al., 2005b; Long et al., 2007; Tao et al., 2010; Guo et al., 2016; Shen et al., 2017; Wisedchaisri et al., 2019; Xu et al., 2019). We have also seen that even among channels with domain-swapped voltage sensors, the S4 helix apparently moves to different extents to regulate the pore (Guo et al., 2016; Shen et al., 2017; Wisedchaisri et al., 2019; Xu et al., 2019). Channels with non-domain-swapped voltage sensors, because they do not have an S4-S5 linker helix, might undergo conformational changes that are not only different in magnitude, but also in character. Obviously, that KvAP has non-domain-swapped voltage sensors is important to the interpretation of functional data, especially those addressing voltage sensor conformational changes (Cuello et al., 2004; Ruta et al., 2005).

KvAP is an outlier among voltage-gated ion channels, whether domain-swapped or not, in two respects. First, the distribution of positive-charged amino acids on S4 is unique. In other Kv channels the region of S4 that contains gating charge residues – the active region – consists of the strict triplet (RXX)n, where R (arginine) is sometimes replaced by K (lysine) and X stands for a hydrophobic amino acid. More rarely R is replaced by a non-charged polar amino acid, but when this occurs the triplet periodicity almost always persists so that the occurrence of subsequent arginine residues remains in-phase in the sequence. But consider the S4 sequence in KvAP: (RLV)(RLL)(RFL)(RIL)(LII)(SRG)(SKF). Here, we observe a discontinuity between the fourth and fifth arginine residues created by six consecutive uncharged amino acids; this produces a phase shift of the fifth and sixth (lysine) with respect to the first four. The fifth R as well as the following lysine in the sequence are certainly part of the active region of S4, because in the structure the lysine resides inside the gating charge transfer center (Figure 5C), which is the site at the center of the voltage sensor that lowers the energy barrier for charged amino acids as they cross the membrane (Tao et al., 2010). In other voltage-gated ion channels, S4 adopts a 310 helix at the center of the membrane; this secondary structure directs the arginine residues towards the gating charge transfer center. The α to a 310 transition may well occur when the region of S4 containing the first four arginine residues approaches the center of the membrane (because they are distributed with triplet periodicity), but it might not occur in the gap between the fourth and fifth arginine residues. Because triplet periodicity is not maintained over the lower half of S4 in KvAP, it is not entirely clear whether properties learned from other Kv channels apply to KvAP.

The second respect in which KvAP is an outlier concerns the gating charge transfer center. KvAP has the typical constellation of negative-charged amino acids to stabilize the positive charge on arginine or lysine. But instead of the phenylalanine found in other Kv channels (357 out of 360 examined), KvAP contains a leucine. The functional significance of this is also unknown.

Our original expectation that KvAP and Shaker-like Kv channels probably undergo similar voltage sensor conformational changes may be incorrect. The present study shows that the voltage sensors in KvAP, in contrast to Shaker, are not domain-swapped. In separate studies in our lab, we have captured the HCN channel, another non-domain-swapped voltage-gated channel, in both depolarized and hyperpolarized conformations (Lee and MacKinnon, 2017; Lee and MacKinnon, 2019). When depolarized, the voltage sensor in HCN is similar to that in KvAP. But when hyperpolarized, the S4 helix translates toward the cytoplasm and breaks into an L-shape, with the bottom half of S4 forming an interfacial helix. Thus, the voltage sensor in HCN couples to the gate in a fundamentally different manner than in domain-swapped voltage-gated channels studied so far, where the S4-S5 linker forms a permanent interfacial helix (Figure 1C). Whether the allosteric communication between the voltage sensor and pore in KvAP will be similar to that observed in HCN remains to be seen.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Aeropyrum pernix)KvAPsyntheticSynthesized at GeneWiz.
Recombinant DNA reagentpET28aNovagen69864
Strain, strain background (Escherichia coli)BL21(DE3)InvitrogenC600003
Chemical compound, drug2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG)AnatraceNG310
Chemical compound, drugCholesteryl hemisuccinate (CHS)AnatraceCH210
Chemical compound, drugDigitoninSigma-AldrichD141
Antibodyanti-KvAP (mouse monoclonal, Fab)DOI: 10.1038/nature015806E1KvAP teramer: 6E1 Fab = 1: 4.4 molar ratio
Commercial assay or kitTalon metal affinity resinClontech635504
Commercial assay or kitSuperdex 200 Increase 10/300 GLGE Healthcare Life Sciences28990944
Commercial assay or kitR1.2/1.3 400 mesh Au holey carbon gridsQuantifoil1210627
Software, algorithmSerialEMDOI: 10.1016/j.jsb.2005.07.007http://bio3d.colorado.edu/SerialEM
Software, algorithmMotionCor2DOI: 10.1038/nmeth.4193https://msg.ucsf.edu/software
Software, algorithmGctfDOI: 10.1016/j.jsb.2015.11.003https://www.mrc-lmb.cam.ac.uk/kzhang/
Software, algorithmGautomatchotherhttps://www.mrc-lmb.cam.ac.uk/kzhang/
Software, algorithmRELION-3DOI: 10.7554/eLife.18722http://www2.mrc-lmb.cam.ac.uk/relion
Software, algorithmcryoSPARCDOI: 10.1038/nmeth.4169http://www.cryosparc.com
Software, algorithmFrealignXDOI: 10.1016/j.jsb.2013.07.005http://grigoriefflab.janelia.org/frealign
Software, algorithmCOOTDOI: 10.1107/S0907444910007493https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software, algorithmPHENIXDOI: 10.1107/S2059798318006551https://www.phenix-online.org
Software, algorithmUCSF ChimeraDOI: 10.1002/jcc.20084https://www.cgl.ucsf.edu/chimera
Software, algorithmPymolPyMOL Molecular Graphics System, Schrödinger, LLChttp://www.pymol.org
Software, algorithmHOLEDOI: 10.1016/s0263-7855(97)00009-xhttp://www.holeprogram.org

Cloning, expression, and purification

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DNA encoding residues 12–282 of KvAP was subcloned into protein expression vector pET28a (Novagen) between NcoI and BglII restriction endonuclease sites with a thrombin cleavage site between a carboxy-terminal hexahistidine sequence and the channel. Channel protein was expressed in BL21(DE3) cells (Invitrogen). For large scale culture, a single colony was inoculated in LB medium overnight at 37°C and then diluted 1:100 (v:v) into auto-inducing ZYM-5052 medium and further inoculated at 37°C for 24–26 hr (Studier, 2005).

The cell pellet from a 2-liter culture was resuspended in 100 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM KCl, 0.1 mg/ml DNase I, and protease inhibitors including 0.1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 0.1 mg/ml soy trypsin inhibitor, 1 mM benzamidine, 0.1 mg/ml 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and 2 mM phenylmethysulfonyl fluoride (PMSF)) and then passed through a microfluidizer three times before adding lauryl maltose neopentyl glycol/cholesteryl hemisuccinate (LMNG/CHS) to a final concentration of 10 mM/2 mM. After 2 hr of extraction at room temperature, the lysate was clarified by centrifugation at 39,800 x g for 30 min. Supernatant was then applied to a prepacked 4 ml Talon Co2+ affinity column (Clontech) equilibrated in wash buffer (20 mM Tris-HCl pH 8.0, 150 mM KCl, 0.06% Digitonin). Nonspecifically bound protein was washed using 15 mM imidazole added to the above buffer. The channel was eluted with 400 mM imidazole, concentrated using an Amicon Ultra centrifugal filter (MWCO 50 kDa), and then injected onto a Superdex-200 (10/30) column (GE Healthcare) equilibrated with wash buffer. Fractions corresponding to KvAP tetramer (indicated by black ** in Figure 2—figure supplement 1) were pooled and concentrated to ~5 mg/ml, mixed with purified 6E1 Fab at a molar ratio of 1:1.1 (KvAP monomer:6E1 Fab) on ice for 0.5 hr, and loaded onto Superdex-200 column again. Fractions corresponding to KvAP-6E1 Fab complex (indicated by red * in Figure 2—figure supplement 1) were pooled, concentrated to ~6.5 mg/ml and used for cryo-EM grid preparation.

Fab preparation

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Monoclonal antibody 6E1 (mouse immunoglobulin) against KvAP were raised using standard procedures (Harlow, 1989) and purified as previously described (Jiang et al., 2003a). In brief, IgGs from mouse hybridoma cell culture supernatant were purified using a Protein A column (Bio-Rad). Fab fragments were generated by papain proteolysis (Worthington) and purified by Q-Sepharose chromatography (GE Healthcare). The hybridoma cell line has not been authenticated or tested for mycoplasma contamination.

Cryo-EM grid preparation and imaging

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3.5 μl of purified protein was pipetted onto glow-discharged Quantifoil Au 400 mesh, R 1.2/1.3 holey carbon grids (Quantifoil). Grids were blotted for 4 s with a blotting force of 1 and humidity of 100% and flash frozen in liquid-nitrogen-cooled liquid ethane using a FEI Vitrobot Mark IV (FEI).

Grids were then transferred to a FEI Titan Krios electron microscope operating at an acceleration voltage of 300 keV. Images were recorded in an automated fashion on a Gatan K2 Summit detector (Gatan) set to super-resolution mode using SerialEM (Mastronarde, 2005). Specifically, images were recorded at a super-resolution pixel size of 0.514 Å and defocus range of 0.8 to 2.2 μm, for 10 s with a subframe exposure time of 200 ms in a dose of approximately 7.9 electrons per pixel per second (a total accumulated dose of approximately 75 electrons per Å2 over 50 subframes or approximately 1.5 electrons per Å2 per subframe).

Image processing and map calculation

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Dose-fractionated super-resolution images were 2 × 2 down sampled by Fourier cropping for motion correction with MotionCor2 (5 × 5 patches) (Zheng et al., 2017). The parameters of the contrast transfer function were estimated by GCTF (Zhang, 2016). Following motion correction,~5 k particles from a subset of the images were interactively selected using Relion to generate templates representing different views for automated particle selection with gautomatch (https://www.mrc-lmb.cam.ac.uk/kzhang/). The autopicked particles were then subjected to 2D classification in Relion to remove false positives as well as particles belonging to low-abundance classes (Scheres, 2012). Particles belonging to 2D classes with more constant 4 Fabs were used for initial model generation in cryoSPARC (Punjani et al., 2017).

~735 k particle images were selected following 4 rounds of 2D classification in Relion (Scheres, 2012). These particles were separated into eight classes using Relion’s 3D classification algorithm without a mask. All eight classes showed presence of 4 Fabs on the opposite side of the CTD density, although the positions of Fabs are variable. One of the eight classes, accounting ~13% (~97 k particles) of the total particles, showed most continuous densities for the KvAP channel moiety after 3D auto-refine in Relion imposing C4 symmetry. The resulting map has a resolution of ~8 Å before postprocessing, showing clearly non-domain-swapped S4-S5 linker density (Figure 2—figure supplement 5). The refined particle images of this class were subjected to further focused 3D classification masking the KvAP transmembrane region and Fabs while applying C4 symmetry and skipping image alignment. Rotational and translational parameters of particles from two major classes (~67 k particles) of the focused 3D classification were used as the input for further focused refinement masking around the KvAP transmembrane region and Fabs in FrealignX, during which the resolution of the reference map used for alignment was limited to 8 Å (Lyumkis et al., 2013). The final map achieved a resolution of ~5.9 Å as assessed by Fourier shell correlation using the 0.143 cut-off criterion (Figure 2—figure supplement 2) and was sharpened using an isotropic b-factor of −500 Å2. Symmetry expansion of the above 67 k particles in Relion followed by local refinement in cryoSPARC slightly improved the map quality (Scheres, 2012; Punjani et al., 2017).

Model building and refinement

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For model generation, the crystal structure of the isolated voltage sensor (PDB: 1QRS) and a model of the pore from the crystal structure of the full-length KvAP channel (PDB: 1ORQ) were placed into the density. Small rigid body adjustments of helical segments were made, followed by a round of real-space refinement in Phenix (Afonine et al., 2018). The only new model rebuilding at the level of individual amino acids involved four amino acids inside the continuous density connecting S4 to S5. Figures were prepared using PyMOL (Molecular Graphics System, Version 2.2.0 Schrodinger, LLC) and Chimera (Pettersen et al., 2004).

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Decision letter

  1. Kenton J Swartz
    Senior and Reviewing Editor; National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
  2. Gilman Toombes
    Reviewer; National Institute of Neurological Disorders and Strokes, United States
  3. Youxing Jiang
    Reviewer; University of Texas Southwestern Medical Center, United States
  4. Fred Sigworth
    Reviewer; Yale, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

In this study, Tao and MacKinnon determine the cryo-EM structure of KvAP channel in complex with the Fab fragment of a monoclonal antibody. Despite of low resolution around 6 Å, the previously determined high resolution crystal structure of the isolated VSD of KvAP and the pore region of the full-length KvAP structure conform well with the EM map, allowing them to build an accurate model into the density. The structure reveal some unexpected features that distinguish KvAP from most other canonical Kv channels: (1) the channel adopts a non-domain-swapped topology; (2) S4 does not appear to form a 310 helix and has a distinct arrangement of voltage-sensing arginine residues; (3) KvAP contains a Leu instead of a Phe at its gating charge transfer center; (4) the relative position between VSD and pore in KvAP is different from that in both domain-swapped and non-domain-swapped Kv channels; (5) S1 and S5 form extensive contact in KvAP, which is not observed in other Kv channels. These distinct features suggest that KvAP is an outlier among voltage-gated ion channels and its structural mechanism of voltage gating is likely different from that of most canonical voltage-gated K+ channels. KvAP was the channel for which the first structures were solved and thus has been widely used as a model protein for studying voltage gating, the findings of this study become particularly important and may provide explanations for some discrepant results in the studies of voltage-dependent conformational changes. Overall, this is a straightforward and informative study that will valuable for understanding the fundamental mechanisms by which voltage-activated ion channels open and close in response to changes in membrane voltage. The editors think you have addressed all of the issues raised by reviewers and we are delighted to be able to publish your work in eLife.

Decision letter after peer review:

Thank you for submitting your article "Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Kenton Swartz serving as both Reviewing Editor and Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Gilman Toombes (Reviewer #1); Youxing Jiang (Reviewer #2); Fred Sigworth (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As you will see from the comments of the reviewers, all three are enthusiastic about publishing your manuscript in eLife and reviewer #1 has suggestions for improving the work in revision. Please consider their comments and address their points in preparing a revised manuscript.

Reviewer #1:

In 2003 the MacKinnon lab revolutionized our understanding of voltage-gated ion channels with the first x-ray crystallographic structures of KvAP, a voltage-gated potassium channel from a thermophilic bacteria. However, KvAP appeared to be distorted in the full-length crystal structure and despite extensive structural and functional studies, the native structure of KvAP is unknown. In this work, the authors have used modern single-particle cryo-EM techniques to obtain a seemingly native-state structure of KvAP in which the voltage-sensing domain (VSD) and pore domain of each KvAP sub-unit are in direct contact, unlike canonical voltage-gated potassium channels (Kv1-9) in which each VSD nestles against the pore domain of the neighboring sub-unit.

This is an important result, and the article is exceptionally clear and well written. However, there are a few aspects of the study that could benefit from further discussion.

1) Effect of Resolution on Model:

The authors have clearly accounted for the EM map resolution (~6 Å) during modelling, but it would help a general reader to present whatever cross-checks the authors performed. For example, did the authors manually assess map uncertainties by plotting different sigma level surfaces, or use a more automated approach (e.g. Beckers, M., Jakobi, A.J., and Sachse, C. (2019). Thresholding of cryo-EM density maps by false discovery rate control. IUCrJ 6, 18-33.). Is the direct S4-S5 linker clearly visible in the raw unmasked ~8 Å map of Class I, or only after masking and symmetry expansion steps? What is the worst resolution at which the EM map could exclude a domain-swapped linker "smeared out" by C4 averaging of multiple conformations, especially if the inner gate were to be in a mixture of open and inactivated states (subsection “Conformational State”)? Does the direct linker density still jump out even if one incorrectly uses a domain-swapped KvAP model (e.g. Figure 6, Lee et al., 2005) for masking and refinement?

2) Effects of Detergent and Antibodies on Conformation:

The MacKinnon lab has previously shown that the conformation of voltage sensing proteins, and KvAP in particular, can be very sensitive to the composition of the lipid membrane. In addition, although Jiang et al. (2003) elegantly showed that binding of the 6E1 antibody drives the channel into a non-conducting state this may not be the physiological inactivated state. In this KvAP structure the VSD domain seems to be displaced towards the extra-cellular side of the membrane (relative to the pore domain) whether one looks at the position of aromatic residues in the TM helices or PEG-Biotin accessibility. It may seem crazy to think lipid composition or antibody-protein interactions could switch a 6-TM channel between direct and domain-swapped configurations, but the Sobolevsky lab observed exactly such a transition for TRPV6 following a single point mutation in the S4-S5 linker (Singh, A.K., Saotome, K., and Sobolevsky, A.I. (2017). Swapping Of Transmembrane Domains In The Epithelial Calcium Channel TRPV6. BioRxiv 141523.) Thus, it would be helpful for the authors to discuss whether the conditions used to obtain the structure could influence the domain organization.

3) Consistency of Structure with Published Cysteine Cross-Linking Data:

The MacKinnon lab has previously reported that KvAP can tetramerize through introduced cysteine residues into the VSD and pore (S4 117C / S5 169C; Figure 5D, Lee et al., 2005; S1 T47C / PD V183C; Lee et al., 2009, PLoS Biol 7, e1000047.) These residue pairs were quite close in KvAP domain-swapped homology models, but are quite distant in this direct KvAP structure. A membrane-based assay to excluded a domain swapped architecture would be especially valuable to check that these cross-links are indeed non-specific.

Reviewer #2:

In this study, Tao and MacKinnon determine the cryo-EM structure of KvAP channel in complex with the Fab fragment of a monoclonal antibody. Despite of low resolution around 6 Å, the previously determined high resolution crystal structure of the isolated VSD of KvAP and the pore region of the full-length KvAP structure conform well with the EM map, allowing them to build a pretty accurately model into the density. The structure reveal some unexpected features that distinguish KvAP from most other canonical Kv channels: (1) the channel adopts a non-domain-swapped topology; (2) S4 does not appear to form a 310 helix and has a distinct arrangement of voltage-sensing arginine residues; (3) KvAP contains a Leu instead of a Phe at its gating charge transfer center; (4) the relative position between VSD and pore in KvAP is different from that in both domain-swapped and non-domain-swapped Kv channels; (5) S1 and S5 form extensive contact in KvAP, which is not observed in other Kv channels. These distinct features suggest that KvAP is an outlier among voltage-gated ion channels and its structural mechanism of voltage gating is likely different from that of most canonical voltage-gated K+ channels. As KvAP has been widely used as a model protein for studying voltage gating, the findings of this study become particularly important and may provide explanations for some discrepant results in the studies of voltage-dependent conformational changes. In short, this is a simple but informative study that will bring some clarifications to the channel field.

Reviewer #3:

This is an important study that answers questions that surrounded the first X-ray structure of a voltage-gated channel, namely this one. It is particularly satisfying to see the mapping of accessibility and EPR experiments on the new structure.

https://doi.org/10.7554/eLife.52164.sa1

Author response

Reviewer #1:

[…]

1) Effect of Resolution on Model:

The authors have clearly accounted for the EM map resolution (~6 Å) during modelling, but it would help a general reader to present whatever cross-checks the authors performed. For example, did the authors manually assess map uncertainties by plotting different sigma level surfaces, or use a more automated approach (e.g. Beckers, M., Jakobi, A.J., and Sachse, C. (2019). Thresholding of cryo-EM density maps by false discovery rate control. IUCrJ 6, 18-33.). Is the direct S4-S5 linker clearly visible in the raw unmasked ~8 Å map of Class I, or only after masking and symmetry expansion steps? What is the worst resolution at which the EM map could exclude a domain-swapped linker "smeared out" by C4 averaging of multiple conformations, especially if the inner gate were to be in a mixture of open and inactivated states (subsection “Conformational State”)? Does the direct linker density still jump out even if one incorrectly uses a domain-swapped KvAP model (e.g. Figure 6, Lee et al., 2005) for masking and refinement?

Yes, we studied these maps carefully at different contours and at different stages of map refinement. The non-domain-swapped S4-S5 linker is clearly visible in the raw unmasked ~8 Å map of Class I. A new supplementary figure (Figure 2—figure supplement 5) is now added to demonstrate this fact.

In cryo-EM image analysis, as it was carried out here, information from an atomic model never enters into the map calculation (unlike model-guided phase refinement in X-ray crystallographic map calculation). Thus, neither a correct nor incorrect model is used to modify phases. The maps we show are experimental. We showed the maps for the entire molecule in stereo to answer the questions this reviewer raises. There is no doubt about the connectivity of the non-helical S4-S5 linker and about the absence of a helical, domain-swapping S4-S5 linker. Furthermore, we show in stereo the unmodified models of the pore and voltage sensor from crystal structures to emphasize how well the map conforms to them.

2) Effects of Detergent and Antibodies on Conformation:

The MacKinnon lab has previously shown that the conformation of voltage sensing proteins, and KvAP in particular, can be very sensitive to the composition of the lipid membrane. In addition, although Jiang et al. (2003) elegantly showed that binding of the 6E1 antibody drives the channel into a non-conducting state this may not be the physiological inactivated state. In this KvAP structure the VSD domain seems to be displaced towards the extra-cellular side of the membrane (relative to the pore domain) whether one looks at the position of aromatic residues in the TM helices or PEG-Biotin accessibility. It may seem crazy to think lipid composition or antibody-protein interactions could switch a 6-TM channel between direct and domain-swapped configurations, but the Sobolevsky lab observed exactly such a transition for TRPV6 following a single point mutation in the S4-S5 linker (Singh, A.K., Saotome, K., and Sobolevsky, A.I. (2017). Swapping Of Transmembrane Domains In The Epithelial Calcium Channel TRPV6. BioRxiv 141523.) Thus, it would be helpful for the authors to discuss whether the conditions used to obtain the structure could influence the domain organization.

We cannot conclude with certainty what the functional state of this channel is. In fact, no one can ever do that with a structure. Based on comparison to other voltage sensors and to the biotin-avidin accessibility data on KvAP, recapitulated in the paper, we conclude the voltage sensors are depolarized. As we stated, the pore could be activated, inactivated (as we would expect based on electrophysiological studies) or some combination of these states.

We do not think that the voltage sensor is displaced towards the extracellular side abnormally. Please note Figure 5A. Also note that voltage sensor Fabs capture the top of the paddle only upon depolarization, and they associate very rapidly, implying the epitope becomes exposed to solution without hindrance by the membrane (Jiang et al. 2003). We think the structure is in good agreement with these observations. Also, the cryo-EM structure is consistent with independent EPR studies of KvAP in lipid vesicles, shown in the paper.

We think that the interchangeable topologies in mutant TRPV6 channels of a single species is a singular observation and very confusing and the physiological significance unclear. Time will tell what those findings mean. Since such behavior has never been seen anywhere else I do not think our paper is the place to discuss it.

Prior to knowing the existence of Kv channels with non-domain-swapped voltage sensor topology, we modelled KvAP as having a domain-swapped voltage sensor (Lee et al., 2005). This was difficult because it seemed there were not enough amino acids to make S4 and an S4-S5 linker helix. Consequently, S4 had to be very short and the tip of the voltage sensor paddle had to be embedded in the membrane. From the present work we now understand the problem. KvAP has a non-domain-swapped topology. The structure we present here is the first hard evidence for the real topology (i.e. we finally see it).

3) Consistency of Structure with Published Cysteine Cross-Linking Data:

The MacKinnon lab has previously reported that KvAP can tetramerize through introduced cysteine residues into the VSD and pore (S4 117C / S5 169C; Figure 5D, Lee et al., 2005; S1 T47C / PD V183C; Lee et al., 2009, PLoS Biol 7, e1000047.) These residue pairs were quite close in KvAP domain-swapped homology models, but are quite distant in this direct KvAP structure. A membrane-based assay to excluded a domain swapped architecture would be especially valuable to check that these cross-links are indeed non-specific.

Both studies (Lee et al., 2005 and Lee et al., 2009) were done in lipid vesicles, not detergents. It is worth pointing out that in our non-domain-swapped cryo-EM model, the Cα-Cα distance between residue 117 (S4) and 169 (S5) on the same subunit is ~35 Å, whereas distance between 117 and 169 on neighboring subunits is ~42 Å. For the other pair, i.e. residue 47(S1) and 183 (pore), the Cα-Cα distance on the same subunit (20.5 Å) and neighboring subunits (21.1 Å). For both cysteine pairs in both models the distances are too far. Neither model is consistent with these disulfide bridges. Unfortunately, when given enough time, as was the case in the Lee et al. studies, disulfide bridges can occur between cysteine residues that are very far apart in protein structures (Careaga and Falke, 1992). Trapping of distant cysteines is a common occurrence unless the kinetics of bridge formation is monitored and shown to be rapid. We now have a much better data set – a structure that explains that KvAP has non-domain-swapped voltage sensors.

https://doi.org/10.7554/eLife.52164.sa2

Article and author information

Author details

  1. Xiao Tao

    Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Project administration, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9381-7903
  2. Roderick MacKinnon

    Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing—original draft, Writing—review and editing
    For correspondence
    mackinn@mail.rockefeller.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7605-4679

Funding

Howard Hughes Medical Institute

  • Xiao Tao
  • Roderick MacKinnon

National Institutes of Health (GM43949)

  • Xiao Tao
  • Roderick MacKinnon

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

Acknowledgements

We thank Mark Ebrahim and Johanna Sotiris at the Evelyn Gruss Lipper Cryo-EM Resource Center at Rockefeller University for assistance in data collection; thank Dr. Joel Butterwick (Rockefeller University) for advice on protein expression, Dr. Xiaochen Bai (UT Southwestern) for advice on data processing; and members of the MacKinnon lab and Chen lab (Rockefeller University) for assistance. This work was supported in part by GM43949. R.M. is an investigator in the Howard Hughes Medical Institute.

Senior and Reviewing Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Reviewers

  1. Gilman Toombes, National Institute of Neurological Disorders and Strokes, United States
  2. Youxing Jiang, University of Texas Southwestern Medical Center, United States
  3. Fred Sigworth, Yale, United States

Publication history

  1. Received: September 24, 2019
  2. Accepted: November 20, 2019
  3. Accepted Manuscript published: November 22, 2019 (version 1)
  4. Version of Record published: November 28, 2019 (version 2)

Copyright

© 2019, Tao and MacKinnon

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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