Journal: The Journal of Cell Biology

Article Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

doi: 10.1083/jcb.202506015

Figure Lengend Snippet: Effect of Mis12c Mtw1c auto-inhibition on the interaction with CENP-U Ame1 . (A) AF2 structure prediction of the interaction between S. cerevisiae Mis12c Mtw1c and CENP-U Ame1 colored by chain, showing only the Mis12c Mtw1c head 1 domain and residues 1–33 of CENP-U Ame1 predicted to interact with Mis12c Mtw1c . The structure prediction was performed using full-length and wild-type proteins apart from Dsn1, from which Dsn1 residues 1–257 were excluded to avoid Mis12c Mtw1c auto-inhibition. Top panel: Overview of the complex. Lower panel: Details of CENP-U Ame1 residues Asp25 to Thr31 interactions with head 1. Val26, Val27, Ile28, and Phe29 of CENP-U Ame1 engage a hydrophobic pocket on head 1, augmented by hydrogen bonds from Asp25 and Thr31. (B) Superimposition of the AF2 model presented in onto the experimental model of Sc Mis12c Mtw1c (this study). The subunits are colored by chain; Mis12 Mtw1 and Nnf1 are colored identically in both the AF2 model and the experimentally determined model. The apparent steric clash between CENP-U Ame1 residues 1–24 and Dsn1 AI is highlighted. (C) Comparison of the AF2 model of CENP-U Ame1 bound to Sc Mis12c Mtw1c (top panel) and Dsn1 AI of Sc Mis12c Mtw1c (bottom panel). Both CENP-U Ame1 and Dsn1 AI form basic α-helices that engage the acid patch of head 1. (D) CENP-C MIF2 and CENP-U Ame1 –binding sites overlap. (E) SEC elution chromatograms of CENP-QU alone, Knl1c:Mis12c Mtw1c complexes that lack residues 1–444 of Knl1 Spc105 alone (K HB-RWD M), or after reconstitution of the interaction between CENP-QU and K HB-RWD M. (F) Coomassie brilliant blue–stained SDS-PAGE gels of the experiments in E. (G) ITC data for (i) CENP-U Ame1_1-33 interactions with KM, (ii) CENP-U Ame1_1-33 interactions with KM 2E , and (iii) the CENP-U Ame1_1-33 mutant with Ala substitutions of D25, V27, I28, F29, T31 predicted to interact with Mis12c Mtw1c to KM 2E . K D and n values are an average from three experiments (technical repeats). Source data are available for this figure: .

Article Snippet: CENP-U Ame1_1-33 (MDRDTKLAFRLRGSHSRRTDDIDDDVIVFKTPNW-amide), CENP-U Ame1_1-33-Mut (MDRDTKLAFRLRGSHSRRTDDIDDAAAVAKAPNW-amide), and CENP-C Mif2_1–38 (MDYMKLGLKSRKTGIDVKQDIPKDEYSMENIDDFFKDD-amide) peptides were synthesized by Alta BioScience.

Techniques: Inhibition, Comparison, Binding Assay, Staining, SDS Page, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

doi: 10.1083/jcb.202506015

Figure Lengend Snippet: Regulation of Mis12c Mtw1c auto-inhibition by Aurora B kinase and its effect on the interaction with CENP-C Mif2 . (A) Structural alignment of the crystal structure of K. lactis CENP-C Mif2 residues 4–36 ( Kl CENP-C Mif2_4-36 ) as bound to the Kl Mis12c Mtw1c head 1 domain (PDB: 5T59 ) to the experimental model of Sc Mis12c Mtw1c (this study). The models are colored by chain; Mis12 Mtw1 and Nnf1 are colored identically in 5T59 and our experimentally determined model. Kl CENP-C Mif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1 AI . Only Kl CENP-C Mif2_4-36 is shown. (B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12c Mtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-C Mif2 residues 1–63 (CENP-C Mif2_1-63 ) indicated within red boxes. (C) Coomassie brilliant blue–stained SDS-PAGE gels of the chromatograms shown in B. (B and C) K HB-RWD M = full-length Dsn1. K HB-RWD M 2E = full-length Dsn1 containing Dsn1 S240E and Dsn1 S250E phosphomimetic mutations. K HB-RWD M Dsn1Δ257 = Dsn1 residues 1–257 are deleted (Dsn1 Δ1-257 ). K HB-RWD M Dsn1Δ226 = Dsn1 residues 1–226 are deleted (Dsn1 Δ1-226 ). K HB-RWD M Dsn1Δ226-2E = Dsn1 residues 1–226 are deleted and Dsn1 S240E and Dsn1 S250E phosphomimetic mutations (Dsn1 Δ1-226,S240E,S250E ). CENP-C Mif2_1-63 is indicated within a red box. (D) ITC data for (i) CENP-C Mif2_1-38 interactions with KM, and (ii) CENP-C Mif2_1-38 interactions with KM 2E . K D and n values are an average from three experiments (technical repeats). Source data are available for this figure: .

Article Snippet: CENP-U Ame1_1-33 (MDRDTKLAFRLRGSHSRRTDDIDDDVIVFKTPNW-amide), CENP-U Ame1_1-33-Mut (MDRDTKLAFRLRGSHSRRTDDIDDAAAVAKAPNW-amide), and CENP-C Mif2_1–38 (MDYMKLGLKSRKTGIDVKQDIPKDEYSMENIDDFFKDD-amide) peptides were synthesized by Alta BioScience.

Techniques: Inhibition, Binding Assay, Staining, SDS Page