Recognition of centromere‐specific histone Cse4 by the inner kinetochore Okp1‐Ame1 complex

Abstract Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP‐A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP‐A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1‐Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.


Expanded View Figures
to high (green).The peptides used for prediction are given at right.E Overlay of Okp1-Ame1-Cse4 from AF2 with the current Okp1-Ame1-Cse4 crystal structure (X-ray).Only Ame1 (magenta and pink) is shown as an opaque chain for clarity.The gray box marks the Okp1-Ame1 head domain.F Overlay of the Okp1-Ame1-Cse4 structure from cryo-EM (EM) with the current crystal structure.The angle between the head and coiled coil shaft is indicated for the cryo-EM structure.Ame1-Leu195, which is the position at which Ame1 bends in the cryo-EM structure, is annotated.The Okp1-Ame1 head domain is marked as in panel E. Structures were aligned on the Okp1-Ame1 coiled coil shaft.The Nkp1-Nkp2 structure from cryo-EM (NKP1 2-76 ; NKP2 4-84 ) is shown as transparent gray chains.

EMBO reports
Sunbin Deng et al A Pulldown assay showing binding between the truncated Okp1-Ame1 complex used for crystallography (Okp1 125-275l -Ame1 124-231 ) and GST-Cse4 END .B Recombinant proteins used for pulldowns in Fig 3 were tested for their association with GST to determine the level of non-specific binding.Results of a GST pulldown assay are shown.C The minimal Okp1-Ame1 complex used for crystallography was tested for its association with FITC-Cse4 END in a fluorescence polarization experiment.The measured dissociation constant is ~750 nM (see Materials and Methods; n = 3 independent experiments).D GST-Cse4 END and its mutants were tested for Okp1-Ame1 binding.E Full-length mutant Okp1-Ame1 complex (EYAA or I195Y as indicated) was tested for its association with FITC-Cse4 END .F Various Okp1-Ame1 mutants (indicated above) were tested for binding to GST-Cse4 END .G Various Okp1-Ame1 mutants were tested for Cse4 END binding as in panel F.
Source data are available online for this figure.
Figure EV1.Crystal structure of Okp1-Ame1-Cse4 END and flexibility at the Okp1-Ame1 head-coiled-coil joint and Nkp1-Nkp2 position.A The crystal structure of Okp1-Ame1-Cse4 colored as in Fig 1.The green box shows the limits of a single unit cell.B Close-up view of an individual biological protomer.Okp1-Ame1-Cse4 colored as in Fig 1. Neighboring protomers are colored gray.C Cse4 END peptide density from the final refined model (top; 2Fo-Fc) and from the refined model lacking Cse4 END (bottom; Fo-Fc, Cse4 omitted).D The structure of Okp1-Ame1-Cse4 as predicted by AlphaFold 2 (AF2) (Jumper et al, 2021).The model is colored according to confidence score (pLDDT) from low (blue)to high (green).The peptides used for prediction are given at right.E Overlay of Okp1-Ame1-Cse4 from AF2 with the current Okp1-Ame1-Cse4 crystal structure (X-ray).Only Ame1 (magenta and pink) is shown as an opaque chain for clarity.The gray box marks the Okp1-Ame1 head domain.F Overlay of the Okp1-Ame1-Cse4 structure from cryo-EM (EM) with the current crystal structure.The angle between the head and coiled coil shaft is indicated for the cryo-EM structure.Ame1-Leu195, which is the position at which Ame1 bends in the cryo-EM structure, is annotated.The Okp1-Ame1 head domain is marked as in panel E. Structures were aligned on the Okp1-Ame1 coiled coil shaft.The Nkp1-Nkp2 structure from cryo-EM (NKP1 2-76 ; NKP2 4-84 ) is shown as transparent gray chains.

Figure EV2 .
Figure EV2.Protein sequence alignments for Okp1 and Ame1 covering the Cse4 END contacts shown in Fig 2. A, B Asterisks mark the Okp1-Ame1 residues shown as sticks in Fig 2.

Figure EV4 .
Figure EV4.In vivo consequences of Okp1-Ame1 mutations.A Western blot showing expression of Ame1, Okp1, and their mutants in whole cell extracts (TAFprotein A-FLAG tag; anti-Protein A used for detection).B-F Tetrad dissection results as in Fig 4B-D.The mutants tested and the resulting spore genotypes are shown at right.Source data are available online for this figure.