Structural Basis of betaKNL2 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 CENP-A/CENH3. This process depends on KNL2/M18BP1 and CENP-C proteins. In plants, two variants of KNL2, KNL2 and {beta}KNL2, are present. Both possess the conserved SANTA domain, while KNL2 additionally has the centromere-targeting CENPC-k motif. Despite lacking the CENPC-like motif, the plant-specific {beta}KNL2 localizes to centromeres and aids in CENP-A/CENH3 loading. We found that efficient centromeric targeting of {beta}KNL2 requires the SANTA domain and the C-terminal part, while nuclear targeting depends on a conserved C-terminal motif-III. Structural predictions and experimental validations reveal that {beta}KNL2 forms homodimers and interacts with centromeric DNA and KNL2. We confirm that centromeric targeting of {beta}KNL2 depends on KNL2 in a tissue-dependent manner. Our findings provide crucial insights into the unique mechanisms of plant-specific kinetochore assembly, highlighting {beta}KNL2s essential role in this process. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/605747v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@30b331org.highwire.dtl.DTLVardef@14494a3org.highwire.dtl.DTLVardef@17a6336org.highwire.dtl.DTLVardef@7ad8f_HPS_FORMAT_FIGEXP M_FIG C_FIG