Lis1 regulates dynein by sterically blocking its mechanochemical cycle

  1. Katerina Toropova
  2. Sirui Zou
  3. Anthony J Roberts
  4. William B Redwine
  5. Brian S Goodman
  6. Samara L Reck-Peterson  Is a corresponding author
  7. Andres E Leschziner  Is a corresponding author
  1. Harvard University, United States
  2. Harvard Medical School, United States
  3. University of Leeds, United Kingdom
6 figures, 3 videos and 3 tables

Figures

Figure 1 with 2 supplements
The binding of Lis1 to dynein changes the position of dynein's linker domain.

(A) Domain organization of dynein and Lis1 constructs used in this study. Dynein's AAA+ domains are labeled AAA1–6. MTBD: microtubule binding domain; CC: coiled coil; LisH: Lis-homology …

https://doi.org/10.7554/eLife.03372.003
Figure 1—figure supplement 1
Three-dimensional (3D) classification and refinement of the dynein and dynein–Lis1 reconstructions.

(A) SDS-PAGE of dynein and Lis1, affinity purified from S. cerevisiae. (B) Comparison between re-projections of the dynein and dynein–Lis1 reconstructions and the best-matching reference-free class …

https://doi.org/10.7554/eLife.03372.004
Figure 1—figure supplement 2
The linker's displaced position in the presence of Lis1 does not appear to involve a specific interaction with AAA4.

(A) Zoomed out view of dynein–Lis1; only the portion of the crystal structure corresponding to AAA4 is displayed, in yellow (PDB ID: 4AKG [Schmidt et al., 2012]). (B) Close-up of the N-terminal …

https://doi.org/10.7554/eLife.03372.005
Figure 2 with 2 supplements
Disrupting the putative dynein–Lis1 interface impairs Lis1's ability to bind to and regulate dynein.

(A) The Lis1 β-propeller engages dynein primarily at a surface helix connecting AAA3 and AAA4 (yellow arrowhead, see Video 3). Inset: a zoomed out view. (B) (Left) View along the axis highlighted in …

https://doi.org/10.7554/eLife.03372.009
Figure 2—figure supplement 1
Probing of the proposed dynein–Lis1 interface by mutagenesis.

(A) Sequence identity (100%, purple; 0%, white) mapped onto the Lis1 homology model. The alignment was carried out with the following species: M. musculus, H. sapiens, S. cerevisiae, A. nidulans, D. …

https://doi.org/10.7554/eLife.03372.010
Figure 2—figure supplement 2
Velocity distributions for dynein alone or in the presence of wild-type or mutant Lis1.

Histogram showing the velocity distribution of single TMR-labeled GST-dynein331kDa molecules in the absence of Lis1 (black) and with 200 nM wild-type Lis1 (light gray), Lis1R378A (medium gray) and …

https://doi.org/10.7554/eLife.03372.011
Figure 3 with 1 supplement
Lis1 sterically blocks the linker domain's normal position on dynein's ring in ADP and no nucleotide conditions but does not prevent it from reaching the pre-powerstroke position at AAA2.

(A) Cryo-NS maps of S. cerevisiae dynein in 100 μM ADP displaying the linker next to either AAA5 (left) or AAA4 (right). The S. cerevisiae linker domain (lacking nucleotide at AAA1, PDB ID: 4AKG [Sch…

https://doi.org/10.7554/eLife.03372.014
Figure 3—figure supplement 1
FRET analysis of linker movement towards the pre-powerstroke position in the presence of Lis1.

(A) Diagram of a microtubule-gliding assay. Monomeric GFP-dynein molecules are immobilized on the coverslip via anti-GFP antibodies (Y shape). Dynein-driven gliding of fluorescently labeled (purple …

https://doi.org/10.7554/eLife.03372.015
Figure 4 with 1 supplement
ATP turnover in the presence of Lis1 requires a hydrolysis-competent AAA1 and a functional AAA5 linker-docking site.

Microtubule-stimulated ATPase activity of dynein monomers carrying (A) wild-type AAA+ modules, (B) a hydrolysis deficient E1849Q mutation in AAA1 (Kon et al., 2004), (C) a hydrolysis deficient …

https://doi.org/10.7554/eLife.03372.016
Figure 4—figure supplement 1
Lis1 binds to dynein ATPase mutants.

(AD) SDS-PAGE of fractions eluted from size-exclusion chromatography runs of Lis1 mixed with each of the dynein constructs used in the ATPase assays. Lis1 co-elutes with all of the constructs.

https://doi.org/10.7554/eLife.03372.017
Figure 5 with 1 supplement
A shortened linker that can physically bypass Lis1 renders dynein Lis1 insensitive.

(A) A short linker construct was designed by docking the crystal structure of the D. discoideum linker (purple ribbon) (PDB ID: 3VKG [Kon et al., 2012]) into our EM map of dynein alone and …

https://doi.org/10.7554/eLife.03372.019
Figure 5—figure supplement 1
The short linker dynein construct shows robust motility, hydrolyzes ATP, and binds Lis1.

(A) Single-molecule motility assays. Kymographs of GST-dimerized full-length and short linker dyneins. Horizontal scale bar = 2 μm, vertical = 30 s. (B) Velocity and run length for short linker and …

https://doi.org/10.7554/eLife.03372.020
Model for the regulation of dynein by Lis1.

(AG) Current view of dynein's mechanochemical cycle. (A) ATP binding to AAA1 induces the low-affinity conformation in dynein's microtubule-binding domain and (B) release from the microtubule. (C) …

https://doi.org/10.7554/eLife.03372.021

Videos

Video 1
The three-dimensional structure of dynein–Lis1.

The movie shows the 3D reconstruction of dynein in complex with Lis1 with 360° rotation about the Y-axis. After this rotation, the EM density is made transparent to display the docked dynein crystal …

https://doi.org/10.7554/eLife.03372.007
Video 2
The three-dimensional structure of dynein.

The movie shows the 3D reconstruction of dynein alone with 360° rotation about the Y-axis. After this rotation, the EM density is made transparent to display the docked dynein crystal structure …

https://doi.org/10.7554/eLife.03372.008
Video 3
The dynein–Lis1 interface.

The movie shows the 3D reconstruction of dynein–Lis1, with the crystal structure of the dynein motor domain and the Lis1 homology model docked in. After a few frames, the EM density disappears to …

https://doi.org/10.7554/eLife.03372.012

Tables

Table 1

Yeast strains

https://doi.org/10.7554/eLife.03372.006
StrainGenotypeFigure(s)
Reference
RPY753MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1331kDa-gs-DHA, pac1Ä::URA3, ndl1Ä::cgLEU2Figure 2, Figure 2—figure supplement 1,2, Figure 5—figure supplement 1
Huang et al., 2012
RPY816MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigures 1–5, Figure 2—figure supplement 1,2, Figure 1—figure supplement 1, Figure 4—figure supplement 1, Figure 5—figure supplement 1
Julie Huang, Harvard Medical School
RPY842MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1-g-1xFLAG-ga-SNAP-KanR, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigures 3,5, Figure 3—figure supplement 1, Figure 5—figure supplement 1
Huang et al., 2012
RPY844MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa, pac1Ä::HygroRFigures 1,4, Figure 1—figure supplement 1, Figure 3—figure supplement 1
Huang et al., 2012
RPY1198MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa-gs-DHA-KanR, pac1Ä::HygroRFigure 5, Figure 5—figure supplement 1
Huang et al., 2012
RPY1245MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3Figure 2
Jeff Moore, University of Colorado
RPY1248MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, dyn1Ä::URA3Figure 2
This work
RPY1302MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1331kDa, pac1Ä::HygroRFigures 1,3
This work
RPY1400MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDa-L2441ybbR, pac1Ä::HygroRFigure 3, Figure 3—figure supplement 1
This work
RPY1422MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-DYN1314kDa-gs-DHA, pac1Ä::HygroRFigures 4,5, Figure 4—figure supplement 1, Figure 5—figure supplement 1
This work
RPY1436MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev- DYN1314kDa, pac1Ä::HygroRFigure 5
This work
RPY1439MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1314 kDa-gs-DHA-KanR, pac1Ä:URA3, ndl1Ä::cgLEU2Figure 5—figure supplement 1
This work
RPY1509MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1331kDa-gs-DHA-KanR, pac1Ä::HygroRFigure 5—figure supplement 1
This work
RPY1510MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-DYN1314kDa-gs-DHA-KanR, pac1Ä::HygroRFigure 5—figure supplement 1
This work
RPY1523MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä3, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, pac1Ä::URA3Figure 2
This work
RPY1524MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, PAC1R378AFigure 2
This work
RPY1525MATa, ura3-52, lys2-801, leu2-Ä1, his3-Ä200, trp1-Ä63, SPC110-GFP::TRP1, HXT1-tdTomato::HIS3, PAC1R275A,R301A,R378A,W419A,K437AFigure 2
This work
RPY1543MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R275A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2—figure supplement 1
This work
RPY1544MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R378A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2, Figure 2—figure supplement 1,2
This work
RPY1545MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1W419A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2—figure supplement 1
This work
RPY1546MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1K437A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2—figure supplement 1
This work
RPY1547MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1 R275A,R301A,R378A,W419A,K437A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2, Figure 2—figure supplement 1,2
This work
RPY1548MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-PAC1R301A, dyn1Ä::cgLEU2, ndl1Ä::HygroRFigure 2—figure supplement 1
This work
RPY1553MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaE1849Q, pac1Ä::HygroRFigure 4, Figure 4—figure supplement 1
This work
RPY1554MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaE2819Q, pac1Ä::HygroRFigure 4, Figure 4—figure supplement 1
This work
RPY1555MATa, his3-11,15, ura3-52, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-DYN1314kDaK3438E,R3445E,F3446D-gs-DHA, pac1Ä::HygroRFigure 4—figure supplement 1, Figure 5—figure supplement 1
This work
RPY1557MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PAC11-13xMYC-TRP1, PGAL1-ZZ-Tev-GFP-3xHA-DYN1331kDaK3438E,R3445E,F3446D-gs-DHA-KanR, pac1Ä::HygroRFigure 4, Figure 4—figure supplement 1
This work
RPY1623MATa, his3-11,15, ura3-1, leu2-3,112, ade2-1, trp1-1, pep4Ä::HIS5, prb1Ä, PGAL1-ZZ-Tev-GFP-3xHA-GST- DYN1331kDaR2857A,N2858A,K2859A,R2861A,S2862A-gs-DHA, pac1Ä::URA3, ndl1Ä::cgLEU2Figure 1—figure supplement 2
This work
  1. DYN1, PAC11, PAC1, and NDL1 encode the dynein heavy chain, dynein intermediate chain, Lis1 and Nudel orthologs, respectively. DHA, SNAP, and ybbR refer to the HaloTag (Promega), SNAP-tag (NEB), and ybbR tag (Yin et al., 2005), respectively. TEV indicates a Tev protease cleavage site. PGAL1 denotes the galactose promoter, which was used for inducing strong expression of Lis1 and dynein motor domain constructs. Genes encoding proteases Pep4 and Prb1 were deleted as noted. Amino acid spacers are indicated by g (glycine), ga (glycine-alanine), and gs (glycine-serine).

Table 2

Dynein:Lis1 ratios in complexes purified by size-exclusion chromatography

https://doi.org/10.7554/eLife.03372.013
GST-dynein331kDaLis1Lis1 (normalized to WT ratio)
WT Lis10.82 ± 0.010.18 ± 0.011.00
Lis1R275A0.85 ± 0.010.15 ± 0.010.80
Lis1R301A0.88 ± 0.010.12 ± 0.010.62
Lis1R378A1.00 ± 0.000.00 ± 0.000.00
Lis1W419A1.00 ± 0.000.00 ± 0.000.00
Lis1K437A0.85 ± 0.010.15 ± 0.010.80
Lis15A1.00 ± 0.000.00 ± 0.000.00
  1. In relation to Figure 2 and Figure 2—figure supplement 1. Fractions were run on SDS-PAGE, stained with SYPRO red, and the bands corresponding to GST-dynein331kDa and wild-type/mutant Lis1 were quantified using ImageJ. The quantification was done using three adjacent lanes corresponding to the peak from size-exclusion. Values are averages of the three lanes ± SD. The ratio for each mutant normalized against that of wild-type Lis1 is also shown.

Table 3

ATPase assay rate measurements

https://doi.org/10.7554/eLife.03372.018
SampleKm(MT)(ìM)kbasal(Motor domain−1.s−1)kcat(Motor domain−1.s−1)
Full-length linker1.06 ± 0.163.51 ± 0.3116.75 ± 0.49
+Lis11.09 ± 0.204.36 ± 0.3015.06 ± 0.49
Short linker0.92 ± 0.104.45 ± 0.2216.98 ± 0.32
+Lis12.05 ± 0.447.14 ± 0.2116.12 ± 0.61
Full-length linker, AAA4 ATPase mutant (E2819Q)1.55 ± 0.144.53 ± 0.1718.80 ± 0.38
+Lis11.10 ± 0.154.60 ± 0.1913.93 ± 0.31
  1. Data were fit to the following equation: kobs = (kcatkbasal) − [MT]/(Km(MT) + [MT]) + kbasal. Km(MT) is the microtubule concentration that gives half-maximal activation. Values are the averages of triplicate readings ± SE of the fit.

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