Molecular basis for inner kinetochore configuration through RWD domain–peptide interactions. (18th October 2017)

• Record Type: Journal Article
• Authors: Schmitzberger, Florian; Richter, Magdalena M; Gordiyenko, Yuliya; Robinson, Carol V; Dadlez, Michał; Westermann, Stefan
• Is Part Of: EMBO journal. Volume 36:Number 23(2017)
• Page Start: 3458
• Page End: 3482
• Publication Date: 2017-10-18
• Subjects: CCAN -- COMA -- kinetochore -- RWD domain -- structural biology; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.201796636
• Languages: English
• ISSNs: 0261-4189

Abstract

Abstract: Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi‐protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies—COMA—consists of subunits Ame1 CENP‐U, Ctf19 CENP‐P, Mcm21 CENP‐O and Okp1 CENP‐Q . A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen‐deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi‐segmented nexus with distinct binding sites for Ame1, Nkp1‐Nkp2 and Ctf19‐Mcm21. Our crystal structure of the Ctf19‐Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19‐Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19‐Mcm21 and Chl4 CENP‐N ‐Iml3 CENP‐L . Absence of this motif results in dependence on the mitotic checkpoint for viability. Synopsis: Integrated molecular and structural characterization of the budding yeast four‐subunit kinetochore complex COMA reveals the CENP‐Q homolog Okp1 as a central organizer of constitutive centromere‐associated network (CCAN) assembly. Mass spectrometry defines subunit topology of COMA bound to inner kinetochore proteins Nkp1‐Nkp2. H/D exchange experiments map contact sites in COMA‐Nkp1‐Nkp2. Crystal structure reveals the contacts of Ctf19‐Mcm21 RWD domains with Okp1. Ctf19‐Mcm21 binding motif of Okp1 tethers Ctf19‐Mcm21 and Chl4‐Iml3 to kinetochores in vivo . Lack of the Okp1 tethering motif makes yeast viability dependent on the mitotic checkpoint. Abstract : Integrated molecular and structural characterization of the yeast COMA complex reveals the CENP‐Q homolog Okp1 as a central organizer of constitutive centromere‐associated network (CCAN) assembly.