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Molecular basis for inner kinetochore configuration through RWD domainâpeptide interactions
Abstract Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multiâprotein assemblies that are evolutionarily conserved between yeasts and humans. One of these assembliesâCOMAâconsists of subunits Ame1CENPâU, Ctf19CENPâP, Mcm21CENPâO and Okp1CENPâQ. 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 hydrogenâdeuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multiâsegmented nexus with distinct binding sites for Ame1, Nkp1âNkp2 and Ctf19âMcm21. Our crystal structure of the Ctf19âMcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19âMcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19âMcm21 and Chl4CENPâNâIml3CENPâL. Absence of this motif results in dependence on the mitotic checkpoint for viability