Volume 26, Issue 17, Pages 2370-2378 (September 2016) CCAN Assembly Configures Composite Binding Interfaces to Promote Cross-Linking of Ndc80 Complexes at the Kinetochore  Gülsah Pekgöz Altunkaya, Francesca Malvezzi, Zuzana Demianova, Tomasz Zimniak, Gabriele Litos, Florian Weissmann, Karl Mechtler, Franz Herzog, Stefan Westermann  Current Biology  Volume 26, Issue 17, Pages 2370-2378 (September 2016) DOI: 10.1016/j.cub.2016.07.005 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 2370-2378DOI: (10.1016/j.cub.2016.07.005) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Budding Yeast Cnn1CENP-T Is Functionally and Physically Linked to the Ctf3 Complex (A) Schematic summary of two different Ndc80 recruitment pathways at the yeast kinetochore. The microtubule-binding interface of the kinetochore is organized by the Mtw1cMis12 complex and Cnn1CENP-T. (B) Quantitative analysis of tetO plasmid segregation by Cnn1ΔHF-TetR in different CCAN subunit deletion backgrounds. Error bars indicate SEM. (C) Comprehensive quantitative analysis of Ame1-associated CCAN subunits in different deletion backgrounds. Quantified kinetochore subunits are displayed on top and bottom. Log10 fold change between respective CCAN deletion mutant and wild-type is indicated by bar graphs. The bait protein Ame1 is highlighted, and deletions that display coordinated behavior have been grouped together. nucl., nucleosome. (D) Diagram summarizing the physical relationships between CCAN subunits in soluble extracts revealed in (C). See also Data S1. Current Biology 2016 26, 2370-2378DOI: (10.1016/j.cub.2016.07.005) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Biochemical Reconstitution Identifies a Stable Five-Subunit Ctf3c-Cnn1-Wip1 Assembly (A) Size-exclusion chromatography (SEC) analysis of the five-subunit Ctf3c-Cnn1-Wip1 complex. Top: SEC elution profile displayed as the trace of A280 (absorbance at 280 nm). Bottom: corresponding Coomassie-stained gel. (B) Cross-link (XL) MS analysis of Ctf3c-Cnn1-Wip1 using the cross-linker Bis(sulfosuccinimidyl) suberate. (C and D) SEC analysis demonstrating interaction between the histone-fold domains of Cnn1HFD-Wip1 expressed in bacteria and Ctf3c expressed in insect cells. Dashed trace indicates marker proteins with molecular weight. (E) CoIP between different Cnn1-Flag constructs and the Ctf3c subunit Mcm16 from lysates of yeast cells arrested in mitosis with nocodazole. See also Figure S1 and Data S2. Current Biology 2016 26, 2370-2378DOI: (10.1016/j.cub.2016.07.005) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 The Conserved C-terminal Extension of the Cnn1 Histone-Fold Domain Is Required for Interaction with the Ctf3 Complex (A) The structure of chicken CENP-T (blue), CENP-W (white), and the extension helices alpha 4 and 5 (green) is depicted according to [6]. Location of the mutated residues is indicated in the structure. Amino acid numbering is indicated for the chicken and the budding yeast CENP-T protein, respectively. (B) Multiple sequence alignment of the histone-fold extension of CENP-T proteins. Three-point mutations are introduced at E346A, L350D, and E351A. HFD, histone-fold domain. (C) Co-expression and purification of 6xHis-Cnn1-Wip1 or 6xHis-Cnn1(mut1)-Wip1, in combination with the Ctf3-StrepII-Mcm16-Mcm22 complex from insect cells. Whole-cell extracts (WCE) and Ni-NTA elutions were separated by SDS-PAGE and analyzed by Coomassie staining. (D) ChIP of Cse4-13xMyc and Cnn1-13xMyc from yeast cells, followed by deep sequencing. Cnn1 and Cse4 are specially enriched at the centromere, as shown by the browser views of chromosome I. (E) The enrichment of Cse4 or Cnn1 at the centromere of chromosome III, IV, XII, and XIV in different cell-cycle stages is shown. The boxed segment denotes the 125 bp yeast core centromere, with the midpoint set at 0 and relative (rel) positions to the midpoint shown on the x axis. The enrichment profiles represent the average from two biological replicates. See also Figure S2. Current Biology 2016 26, 2370-2378DOI: (10.1016/j.cub.2016.07.005) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 The Five-Subunit Ctf3c-Cnn1-Wip1 Assembly Arranges Two Ndc80 Molecules into a Parallel Configuration (A) SEC analysis of Ctf3c-Cnn1Wip1 alone (top), Ndc80c alone (middle), and a combination of Ndc80 complex and Ctf3c-Cnn1-Wip1 complex (bottom). Boxes indicate fractions selected for EM analysis. The shift in the Ndc80c-Ctf3c-Cnn1-Wip1 elution position is observed from the comparison of the Coomassie-stained gels and from the absorption profile of SEC. (B) The SEC elution profiles of three different molar ratios between Ndc80c and Ctf3c-Cnn1-Wip1. (C) Cross-link (XL) MS analysis of Ndc80c bound to Ctf3c-Cnn1-Wip1 using the cross-linker Bis(sulfosuccinimidyl) suberate. Cross-links between Spc24 and the N terminus of Cnn1 are highlighted in the zoomed box. (D and E) Overview and gallery of rotary shadowing EM analysis of Ndc80 complex (D) and Ndc80c+Ctf3c-Cnn1-Wip1 (E) from the indicated peak fractions of SEC analysis. Position of head domains is indicated by orange arrowheads. Scale bars, 50 nm. (F) Comparison of end-to-end length of particles with two visible heads (Ndc80Cc-Ctf3c-Cnn1-Wip1) and one head (Ndc80c alone). Fifty particles were analyzed for each construct. (G) Model for the organization of the Ndc80 complex bound to Ctf3c-Cnn1-Wip1. The position of the N-termini of the subunits Cnn1, Mcm16, and Mcm22 is indicated. See also Figure S3 and Data S3. Current Biology 2016 26, 2370-2378DOI: (10.1016/j.cub.2016.07.005) Copyright © 2016 Elsevier Ltd Terms and Conditions