Structural basis of βKNL2 centromeric targeting mechanism and its role in plant-specific kinetochore assembly

The kinetochore is an essential protein complex that ensures proper chromosome segregation during cell division. Kinetochore assembly is initiated by the incorporation of centromere-specific Histone H3 (CENP-A/CENH3) into centromeric nucleosomes. This process depends on KNL2/M18BP1 and CENP-C proteins. In eudicots, two variants of KNL2 are present, namely αKNL2 and βKNL2. Both possess the conserved SANTA domain, while αKNL2 additionally has the centromere-targeting CENPC-k motif. Despite lacking the CENPC-like motif, the plant-specific βKNL2 localizes to centromeres and aids in CENP-A/CENH3 loading. We found that efficient centromeric targeting of βKNL2 requires the SANTA domain and the C-terminal part, while nuclear localization is regulated by a conserved C-terminal motif-III, which undergoes SUMOylation. Independent experiments supported by structural analysis suggest that βKNL2 can interact multivalently with αKNL2, with DNA, and itself. We show that the centromeric targeting of βKNL2 depends on αKNL2 in a tissue-dependent manner. Our findings provide crucial insights into the unique mechanisms of plant-specific kinetochore assembly, highlighting βKNL2's essential role in this process.