Abstract
The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. CENP-C (centromere protein C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2CENP-C cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between S. cerevisiae (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution requires investigation. Here, we report the crystal structures of the Schizosaccharomyces pombe and Drosophila melanogaster CENP-C cupin domains at 2.52 and 1.81 Å resolutions, respectively. Although the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, we found that the S. pombe Cnp3CENP-C jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, our results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor.
Highlights
The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle
To elucidate the structural role of the cupin fold and map the binding region required for Moa1 recruitment to centromeres, we introduced 10 point mutations within the SpCnp3CENP-C cupin domain that can be broadly classified into two categories: those within the pocket (N572S, K546D, M574T, A552T, H624A, and F541A) and those on the surface (Y607C, S609R, T586A, and V566A) of the cupin domain (Fig. 7A)
Along with the previously published crystal structure of the S. cerevisiae Mif2CENP-C cupin domain, these three structures originate from organisms with diverse centromere architecture and kinetochore composition
Summary
The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. Structures of CENP-C cupin domains at regional centromeres and subsequently recruiting other CCAN components to the centromere [12,13,14,15,16]. By comparing CENP-C cupin domain structures from organisms with point centromeres (S. cerevisiae) against those with regional centromeres and differing kinetochore compositions (S. pombe and D. melanogaster), we sought to delineate the conserved and unique structural features among cupin domains and further elucidate their functional relevance in kinetochore assembly. A former yeast two-hybrid study has demonstrated that the S. pombe Cnp3CENP-C (hereafter referred to as SpCnp3CENP-C) cupin domain interacts with Moa, a key meiosis I–specific protein and functional equivalent of human MEIKIN, and recruits it to centromeres [23, 27, 28]. Our results further reveal a structural role for the SpCnp3CENP-C C terminus in meiosis
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