Cnp1 N-terminal dynamics regulate L1 loop recognition by Mis15 to orchestrate kinetochore assembly in Schizosaccharomyces pombe.

Centromeres are defined by the histone H3 variant CENP-A, which serves as the foundation for kinetochore assembly to ensure faithful chromosome segregation. CENP-A nucleosomes possess distinctive dynamic features, including flexible DNA ends at the entry/exit sites and a mobile N-terminal region, which are properties proposed to facilitate kinetochore assembly, yet the underlying molecular mechanisms remain elusive. Here, we present cryo-electron microscopy structures of Cnp1-the Schizosaccharomyces pombe ortholog of CENP-A-nucleosomes alone and in complex with Mis15, the fission yeast ortholog of CENP-N. By integrating structural, biochemical, and molecular dynamics analyses, we demonstrate that the N-terminal region of Cnp1 regulates both DNA-end breathing and the conformational mobility of the L1 loop, a critical structural element for Mis15 recognition. Either enhanced dynamics caused by N-terminal deletion or reduced dynamics from targeted residue substitution disrupt Mis15 binding in vitro and impair its centromeric localization in vivo, thereby compromising the earliest steps of constitutive centromere-associated network assembly. Our findings establish the Cnp1 N-terminus as a dynamic allosteric modulator of chromatin architecture and reveal an L1 loop modulation mechanism that links nucleosome flexibility to kinetochore specification and chromosome segregation fidelity in fission yeast.