Structural basis for centromere maintenance by Drosophila CENP‐A chaperone CAL1

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP‐A‐containing nucleosomes. To preserve centromere identity, CENP‐A must be escorted to centromeres by a CENP‐A‐specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP‐A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X‐ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP‐A histone chaperone, binds both CENP‐A and the centromere receptor CENP‐C without the requirement for the Mis18 complex. While an N‐terminal CAL1 fragment wraps around CENP‐A/H4 through multiple physical contacts, a C‐terminal CAL1 fragment directly binds a CENP‐C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP‐A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP‐C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP‐C dimer. Overall, by demonstrating how CAL1 binds CENP‐A/H4 and CENP‐C, we provide key insights into the minimalistic principles underlying centromere maintenance. Synopsis Epigenetic maintenance of centromeres requires specific deposition of histone H3 variant CENP‐A. X‐ray crystallography explains how the evolutionary distinct histone chaperone CAL1 combines both CENP‐A recognition and centromere targeting activities of the respective mammalian counterparts, HJURP and Mis18 complex. Crystal structures of Drosophila CAL1 bound to CENP‐A/H4 dimers show that CAL1 employs evolutionarily conserved and adaptive interactions to recognise CENP‐A/H4. CAL1 binding shields key CENP‐A/H4 surfaces required for nucleosome assembly, a key requirement for preventing untimely CENP‐A loading. CAL1 interactions with its centromeric receptor CENP‐C involve a short helical CAL1 segment spanning residues 890–913, and the CENP‐C cupin domain. CAL1 binds CENP‐C at a site stabilised by cupin domain dimerization, thereby restricting binding to one CAL1 molecule per CENP‐C dimer. Graphical Abstract X‐ray crystallography reveals how the evolutionary distinct histone chaperone CAL1 binds both pre‐nucleosomal CENP‐A/H4 histone dimers and the centromere receptor CENP‐C, thus combining the ac