Structural basis for microtubule recognition by the human kinetochore Ska complex

Abstract

The ability of kinetochores (KTs) to maintain stable attachments to dynamic microtubule structures ('straight' during microtubule polymerization and 'curved' during microtubule depolymerization) is an essential requirement for accurate chromosome segregation. Here we show that the kinetochore-associated Ska complex interacts with tubulin monomers via the carboxy-terminal winged-helix domain of Ska1, providing the structural basis for the ability to bind both straight and curved microtubule structures. This contrasts with the Ndc80 complex, which binds straight microtubules by recognizing the dimeric interface of tubulin. The Ska1 microtubule-binding domain interacts with tubulins using multiple contact sites that allow the Ska complex to bind microtubules in multiple modes. Disrupting either the flexibility or the tubulin contact sites of the Ska1 microtubule-binding domain perturbs normal mitotic progression, explaining the critical role of the Ska complex in maintaining a firm grip on dynamic microtubules.

Figure 1. Characterization of functional determinants of Ska1.

(a) Limited proteolysis of the Ska1_92–255 with trypsin that led to the formation of a stable fragment identified by MS as Ska1_133–255. Uncropped scan of the gel is shown in Supplementary Fig. S8a. (b) Top, representative SDS–PAGE of cosedimentation assays comparing the MT-binding activity of Ska1_133–255 and GST–Ska1_133–255. Bottom, quantification of the MT-binding assays in b (mean±s.d., n=4, ***P0.001, t-test). (c) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from two or more independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time in mitosis (from NEBD until anaphase onset/cell death) and the percentage of cells dying in mitosis. (d) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in c. Time in h:min is indicated. T=0 was defined as the time point at which NEBD became evident. Scale bar, 10 μm. (e) Cartoon representation of the structure of human Ska1–MTBD, which possesses a modified winged-helix domain with an elongated shape. The length of the structure is ~50 Å whereas the width is ~30 Å. Secondary structure elements are labelled. (f) Sequence alignment of human Ska1_91–255 showing amino acid conservation between H. sapiens (hs), Mus musculus (mm), Xenopus tropicalis (xt) and Danio rerio (dr). Secondary structure elements are shown below the aligned sequences. Amino acid conservation is highlighted in grey.

Figure 2. The Ska complex binds MTs through multiple positively charged clusters.

(a) Cartoon representation of the Ska1–MTBD where surface-exposed K/R residues are shown as sticks (left). Surface representation of the Ska1–MTBD in the same orientation with electrostatic surface potential revealing the presence of positively charged patches (right). Residues clustered based on their proximity and mutated to A to test their involvement in MT recognition are highlighted in different colours. (b) Cosedimentation assays of the different K/R- to A-untagged Ska mutants were performed. Representative gels (upper panel) and quantifications of MT cosedimentation assays (bottom panel). Concentration of Ska1 mutants and MTs used in the assays are 3 μM and 6 μM, respectively (mean±s.d., n3, *P0.05, ***P0.001; t-test; right bottom panel). K_d values were calculated using 1 μM Ska and 0–12 μM MTs (bottom left panel). Uncropped scans of the gels are shown in Supplementary Fig. S8b.

Figure 3. MT recognition of Ska1 via its multipartite mode of MT binding is a functional requirement for the Ska complex.

(a) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from two or more independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time in mitosis (from NEBD until anaphase onset/cell death) and the percentage of cells dying in mitosis. (b) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP. Scale bar, 10 μm.

Figure 4. Ska1 interacts with MTs by recognizing the globular regions of tubulin monomers in multiple orientations.

(a,b) Linkage map showing the sequence position of all the crosslinked residue pairs between (a) Ska1–MTBD (b) Ska complex and α1B and β2B tubulin, where 6 μM human Ska complex/Ska1–MTBD was incubated with 10 μM of MTs in the crosslinking reactions. Crosslinked products were resolved in SDS–PAGE followed by MS analysis. Red and green boxes above the β-tubulin show regions of tubulin involved in longitudinal and lateral contacts, respectively. Crosslinks observed between Ska1 R155/236/245K mutant and MTs are shown in grey. (c) Cartoon representation of tubulin dimer where residues involved in crosslinking with Ska1 residues are highlighted in stick representation. Grey and green lines denote crosslinks observed between K/R clusters of Ska1 and Glu/Asp/Tyr/Thr clusters of β-tubulin and α–tubulin, respectively. The region where Ndc80 interacts with tubulin as reported in Alushin et al. is shown in yellow. Important Ska1 residues involved in MT binding are colour coded as in Figs 2 and 3.

Figure 5. Ska and Ndc80 complexes recognize different structural features of MTs.

(a) Left, representative SDS–PAGE. Right, quantification of MT cosedimentation assays with Ndc80 and Ska complexes (1 μM) binding to taxol-stabilized MTs or curved protofilaments induced by vinblastine (6 μM). (b) Quantifications of MT cosedimentation assays with 3 μM Ska and 6 μM vinblastine spirals (right panel) (mean±s.d., n3, ***P0.001; t-test). K_d values were calculated using 1 μM Ska and 0–12 μM vinblastine spirals (left panel). (c) Left, representative SDS–PAGE. Right, quantification of MT cosedimentation assays with Ndc80 and Ska complexes (1 μM) binding to MTs or subtilisin-treated MTs (6 μM; mean±s.d., n3, ***P0.001; t-test). Uncropped scans of the gels are shown in Supplementary Fig. S8c.

Figure 6. Constitutive phosphorylation of S185/T205 perturbs MT binding of the Ska1–MTBD in vitro.

(a) Cartoon representation of Ska1–MTBD, where consensus Aurora B phosphorylation sites are shown in stick representation (red). The phosphorylation sites of Aurora B are located within two of the three main clusters responsible for the MT-binding activity of Ska1 (circled). (b) Representative SDS–PAGE of the MT cosedimentation assays where phosphomimic mutants S185D and S185/T205D are tested. (c,d) Left, schematic representation of both protocols used for the treatment of Ska or Ndc80 complex with Aurora B. Middle, representative SDS–PAGE of the cosedimentation assays, where (c) the Ska/Ndc80 complex was incubated first with Aurora B kinase and subsequently with MTs. (d) MT-bound Ska/Ndc80 complex was incubated with Aurora B. Right, quantification of the results obtained (mean±s.d., n3, *P0.05, ***P0.001; t-test). Uncropped scans of the gels are shown in Supplementary Fig. S8d.

Figure 7. Schematic model.

Schematic model summarizing the mode of MT binding of Ska1 and its implications for maintaining stable KT–MT attachments.