CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation

  1. Axel F Brilot
  2. Andrew S Lyon
  3. Alex Zelter
  4. Shruthi Viswanath
  5. Alison Maxwell
  6. Michael J MacCoss
  7. Eric G Muller
  8. Andrej Sali
  9. Trisha N Davis
  10. David A Agard  Is a corresponding author
  1. Department of Biochemistry and Biophysics, University of California at San Francisco, United States
  2. Department of Biochemistry, University of Washington, United States
  3. Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, United States
  4. Department of Genome Sciences, University of Washington, United States
8 figures, 1 table and 3 additional files

Figures

Spc110p and γ-tubulin small complexes (γTuSCs) assemble to form γ-tubulin ring complexes (γTuRCs) prior to microtubule nucleation.

(A) An overview of the effect of Spc110p deletions on assembly and viability, summarizing previously published data from Lyon et al., 2016. Assembly data was generated using Spc110p-GCN4 tetramer …

Figure 2 with 4 supplements
The Spc110pNCC binds near the N-terminus of Spc97p.

(A) Spc110p N-terminal region secondary structure prediction, showing lack of predicted secondary structure for the first 111 residues. Also shown are Spc110pCM1(117-146) and the Spc110pNCC(164-208)

Figure 2—figure supplement 1
Overview of crosslinking mass spectrometry (XL-MS) datasets.

XL-MS datasets for γ-tubulin small complex (γTuSC) crosslinked to Spc110p1-220-GCN4 dimer (A, B) or Spc110p1-401-GST (C, D) with either EDC (A, C) or DSS (B, D). Each colored rectangle represents a …

Figure 2—figure supplement 2
Results of integrative modeling of the Spc110p-γ-tubulin small complex (γTuSC) complex.

The modeling results shown are based on the γTuSC-Spc110p1-220 GCN4 crosslinks; similar results were obtained in all cases using γTuSC-Spc110p1-401-GST crosslinks (see Appendix 1). (A) Monomer of …

Figure 2—figure supplement 3
The four stages of integrative modeling of the Spc110-γ-tubulin small complex (γTuSC) complex.

This schematic describes the integrative structure modeling procedures used in this paper. The first row details the information to be used in modeling. The background color of each information …

Figure 2—figure supplement 4
Results for sampling exhaustiveness protocol for modeling the complex of Spc110p1-220-GCN4 dimer with γ-tubulin small complex (γTuSC).

(A) Results of test 1, convergence of the model score, for the 2840 good-scoring models; the scores do not continue to improve as more models are computed essentially independently. The error bar …

Figure 3 with 5 supplements
Structure and assembly interfaces of γTuRCWT and γTuRCSS.

(AB) Segmented density of (A) open γTuRCWT and (B) closed γTuRCSS. γTuRC subunits are colored as in the figure inset. Density was segmented within 4.5 Å of the atomic model, showing one Spc110p …

Figure 3—figure supplement 1
γTuRCWT processing and resolution.

(A) Classification scheme for γTuRCWT processing. Images are projection images of the 3D classes obtained. (B) Fourier shell correlation FSC (blue) and map to model FSC (orange) for the closed γTuRCW…

Figure 3—figure supplement 2
γTuRCSS processing and resolution.

(A) Classification scheme for γTuRCSS processing. Images are projection images of the 3D classes obtained. (B) FSC (blue) and map to model FSC (orange) for the γTuRCSS. γTuRC: γ-tubulin ring complex.

Figure 3—figure supplement 3
γTuRCSS and γTuRCWT local resolution maps.

(A) γTuRCSS local resolution map. The color scale is shown between panels (A) and (B). (B) γTuRCWT local resolution map. The color scale is shown between panels (A) and (B). (C) Local resolution map …

Figure 3—figure supplement 4
Wiring diagrams of (A) Spc97p and (B) Spc98p.

Helices are numbered from 1, whereas sheets are labeled from (A). Turns are labeled by type. Plots were generated using the pdbsum server (Laskowski, 2009).

Figure 3—figure supplement 5
Comparison of γ-tubulin conformation between human and yeast γ-tubulin ring complex (γTuRC).

(A) An alignment of γ-tubulin (γ-tubulin bound to Spc98p, khaki, this work; human free γ-tubulin, sky blue, PDB ID 1Z5W; yeast straight β-tubulin, forest green, PDB ID 5W3F) using their N-terminal …

Figure 4 with 3 supplements
The Spc110p centrosomin motif 1 (CM1) helix binds at the inter-γ-tubulin small complex (γTuSC) interface.

(A) Filtered segmented difference map between experimental density and the fitted atomic model without Spc110p overlaid on a γTuRCSS surface lacking Spc110p. The difference map was segmented to show …

Figure 4—figure supplement 1
Spc110pNCC structure (forest green) near the tyrosine 186 side chain fitted into ~4.2 Å low pass filtered density from the γTuRCSS reconstruction. γTuRC: γ-tubulin ring complex.
Figure 4—figure supplement 2
Helix dipole interactions define the centrosomin motif 1 (CM1) binding site on Spc98p.

(A) View of the Spc110pCM1 motif binding site with Spc98p with the H19-S3 region, colored as in Figure 4. Spc110pCM1:Spc98p hydrogen bond interactions are indicated in red. All of the hydrogen bonds …

Figure 4—figure supplement 3
Conserved binding interface with the centrosomin motif 1 (CM1) motif.

(A) Overview of a dimer of γ-tubulin small complex (γTuSC) colored as in Figure 2, with the central Spc97p/98p colored according to their conservation. (B) View of the Spc110pCM1 binding site at the …

Figure 5 with 7 supplements
Structural overview of γ-tubulin ring complex (γTuRC) assemblies.

Top and side views of open γTuRCWT (A, C) and closed γTuRCSS (B, D). Panels (E) and (F) show a bottom view of an assembled γTuRCSS. The arrow indicates the seventh Spc110pNCC binding site in the …

Figure 5—figure supplement 1
A mixture of compositional states is observed.

(A) A filtered micrograph showing raw particles shows well-dispersed single particles and a mixture of γ-tubulin small complex (γTuSC) monomers and dimers. (B) Representative examples of classes …

Figure 5—figure supplement 2
WT γ-tubulin small complex (γTuSC) processing and resolution.

(A) Classification scheme for WT γTuSC monomer and dimer processing. (B) FSC (blue) and map to model FSC for the closed WT γTuSC monomer. (C) FSC (blue) and map to model FSC for the open WT γTuSC …

Figure 5—figure supplement 3
Segmented single-particle reconstructions of γ-tubulin small complex (γTuSC) monomer and dimer.

(A) Approximately 3.7 Å reconstruction γTuSC showing Spc97p (sky blue), Spc98p (dodger blue), and γ-tubulin (khaki). (B) Approximately 4.5 Å reconstruction of a dimer of γTuSC colored as in panel (A)…

Figure 5—figure supplement 4
Conformational changes in γ-tubulin small complex (γTuSC) during assembly and activation.

(A) Views of the monomeric γTuSC aligned with an open γTuRCWT filament monomer using the N-terminal helical bundles of Spc97/98p. Monomeric γTuSC is colored as in Figure 2, and the γTuSCWT filament …

Figure 5—video 1
Conformational changes of γ-tubulin small complex (γTuSC) during assembly.

Morph of the monomeric γTuSC conformation to the conformation adopted by a γTuRCWT filament monomer in the open state. The γTuSC is colored with Tub4p in khaki, Spc97p in light blue, and Spc98p in …

Figure 5—video 2
Conformational changes of γ-tubulin ring complex (γTuRC) during activation.

Morph of a γTuRCWT filament monomer in the open conformation to the conformation adopted by a γTuRCSS filament monomer in the closed state. The γTuSC is colored with Tub4p in khaki, Spc97p in light …

Figure 5—video 3
Morph of γTuRCSS and γTuRCWT closed states shows minimal changes.

Morph of a γTuRCSS filament monomer in the closed state to the conformation adopted by a γTuRCWT filament monomer in the closed state. The γTuSC is colored with Tub4p in khaki, Spc97p in light blue, …

Phosphorylation sites visualized on the γTuRCSS structure.

(A) γTuSCSS dimer, colored as in Figure 2, with phosphorylation sites from Fong et al., 2018 marked with red balls (no known phenotype) or purple balls (phenotype previously reported). Boxes are …

Figure 7 with 2 supplements
Metazoan γ-tubulin ring complexes (γTuRCs) require large motions to template microtubules.

(A) Yeast closed (this work), (B) Xenopus (PDB ID 6TF9), and (C) human γTuRC (PDB ID 6V6S) structures placed adjacent to a microtubule to illustrate the motions required to properly template …

Figure 7—figure supplement 1
A centrosomin motif 1 (CM1) helix binds between grip-containing protein (GCP)2 and GCP6 in human γ-tubulin ring complex (γTuRC).

(A) Human γTuRC density (EMDB ID 21068) is fitted with the C-terminal region of yeast GCP2, γ-tubulin, and CM1 helix (this work), showing that a CM1 helix binds between GCP2 and GCP6 in human γTuRC. …

Figure 7—figure supplement 2
γ-Tubulin ring complex (γTuRC) undergoes large structural changes on centrosomin motif 1 (CM1) binding.

(A, B) Overlay of the two γ-tubulins adjacent to the CM1 binding site observed in the human γTuRC structure for (A) Xenopus (no CM1 bound) and (B) human (CM1-bound) highlighting the large motion …

Model of γ-tubulin ring complex (γTuRC) assembly and activation.

γ-Tubulin small complex (γTuSC) monomers bound to Spc110p display an improved binding for γTuSC due to the presence of the overhanging Spc110pCM1 binding surface. This leads to cooperative assembly …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Software, algorithmIMP (Integrative Modeling Platform)https://integrativemodeling.org;https://doi.org/10.1371/journal.pbio.1001244RRID:SCR_002982Version 2.8
Software, algorithmUCSF Chimerahttps://www.cgl.ucsf.edu/chimera/ https://doi.org/10.1002/jcc.20084RRID:SCR_004097
Strain, strain background (Escherichia coli)BL21(DE3) CodonPlus-RILAgilentPart No.:230245
Genetic reagent (Homo sapiens, Saccharomyces cerevisiae)pET28a-3C-Xrcc4-Spc110(164-207)This paperUniprot:Q13426 (Xrcc4); Uniprot:32380 (Spc110)Construct contains residues 2–132 of H. sapiens Xrcc4 fused with residues 164–207 of S. cerevisiae Spc110
Software, algorithmXDSKabsch, 2010; DOI: https://doi.org/10.1107/S0907444909047337RRID:SCR_015652Version: October 15, 2015
Software, algorithmPhenixAdams et al., 2010; DOI: https://doi.org/10.1107/S0907444909052925; McCoy et al., 2007; DOI: https://doi.org/10.1107/S0021889807021206; Terwilliger et al., 2008; DOI: https://doi.org/10.1107/S090744490705024X Afonine et al., 2012; DOI:https://doi.org/10.1107/S0907444912001308RRID:SCR_014224Version 1.10.1_2155
Software, algorithmCootEmsley et al., 2010; DOI: https://doi.org/10.1107/S0907444910007493RRID:SCR_014222Version 0.8.3
Software, algorithmKojak, XL identification algorithmhttp://www.kojak-ms.org/RRID:SCR_021028Versions 1.4.1 and 1.4.3
Software, algorithmProXL, protein XL data visualizationhttps://proxl-ms.org/RRID:SCR_021027
Chemical compound, drugDSSThermo Fisher Scientific21655
Chemical compound, drugEDCThermo Fisher ScientificA35391
Chemical compound, drugSulfo-NHSThermo Fisher ScientificA39269
Software, algorithmcisTEMGrant et al., 2018.DOI:10.7554/eLife.35383RRID:SCR_016502Version 1.0 beta
Software, algorithmRelionScheres, 2012. PMID:23000701; DOI: 10.1016/j.jsb.2012.09.006 RRID:SCR_016274
Genetic reagent (S. cerevisiae)pFastBac-Tub4pVinh et al., 2002. doi: 10.1091/mbc.02-01-0607
Genetic reagent (S. cerevisiae)pFastBac-Spc97pVinh et al., 2002. doi: 10.1091/mbc.02-01-0607
Genetic reagent (S. cerevisiae)pFastBac-Spc98pVinh et al., 2002. doi: 10.1091/mbc.02-01-0607
Genetic reagent (S. cerevisiae)pFastBac-GST-Spc110p1-220Vinh et al., 2002. doi: 10.1091/mbc.02-01-0607
Genetic reagent (S. cerevisiae)pFastBac-Tub4pS58C/G288CKollman et al., 2015. DOI: 10.1038/nsmb.2953

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