Structural Characterization of CENP-C Cupin Domains at Regional Centromeres Reveals Unique Patterns of Dimerization and Functions for the Inner Pocket

Abstract

Cell division is vital to the development and well-being of all living organisms. This process must occur without error and depends on the equal and accurate division of genetic material to daughter cells. If chromosomes fail to segregate properly, the consequences are often severe and can include cell death, birth defects, and cancer. The centromere and kinetochore are two factors that are required for the successful completion of cell division. The centromere is a unique chromosomal region that is required for specifying the location of kinetochore assembly. In turn, the kinetochore, a multi-protein complex, assembles onto the centromere during cell division and facilitates the formation of functional microtubule attachments. Interestingly, while the functions of both the centromere and kinetochore are highly conserved throughout evolution, their underlying organization and composition vary greatly between organisms. Saccharomyces cerevisiae possess unique point centromeres that are genetically defined by a 125 bp DNA sequence. On the other hand, the majority of other eukaryotes possess much larger regional centromeres whose locations are epigenetically specified by the histone H3 variant, CENP-A. As each organism has its own unique centromere and kinetochore composition, their kinetochore proteins likely possess differing mechanisms of recruitment and function. Consequently, their respective kinetochore proteins may possess variations in structure to accommodate these differences. CENP-C is a particularly interesting inner kinetochore component to study due to its evolutionary conservation and scaffolding roles that connect the inner and outer kinetochores. At its C-terminus, CENP-C harbors a conserved cupin domain that has an established role in CENP-C homodimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2 cupin domain has been determined, it is not yet known whether this domain is structurally conserved within organisms with regional centromeres. Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution, requires investigation. This dissertation focuses on the structural conservation of the CENP-C cupin domain and elucidating its functional significance beyond dimerization. Here, I report the crystal structures of two CENP-C cupin domains from organisms with regional centromeres, Schizosaccharomyces pombe and Drosophila melanogaster. While 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 to facilitate dimerization. In addition, analysis of the Schizosaccharomyces pombe Cnp3 cupin domain in vitro and in vivo shows the inner pocket formed by its jelly roll fold functions as a binding surface for the meiosis-specific protein, Moa1. Thus, these results unveil the evolutionarily conserved and novel features of the CENP-C cupin domain, as well as its additional role as a recruitment factor.