Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein–dynactin adaptor Spindly and the ROD–Zwilch–ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high‐resolution cryo‐EM structure that captures the essential features of the RZZ complex, including a farnesyl‐binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ–Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation‐dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long‐sought‐for molecular basis of corona assembly on metazoan kinetochores. Synopsis The structure of the ROD‐Zwilch‐ZW10 (RZZ) complex reveals the organisation of the building block of a polymeric fibrous structure of the kinetochore, the corona. Determinants of corona assembly are identified by in vitro reconstitution and structural dissection and validated in cell biology experiments. The cryo‐EM structure of the RZZ complex, the main constituent of the kinetochore corona, is reported. Polymerization of the corona is recapitulated in vitro with purified components. The dynein‐dynactin adaptor Spindly and MPS1 phosphorylation drive RZZ corona assembly. A farnesyl‐binding site in the ROD subunit promotes Spindly binding. A kinetochore binding domain of RZZ:Spindly is identified. Graphical Abstract The cryo‐EM structure of the ROD‐Zwilch‐ZW10 complex provides insight into the organization and assembly of the kinetochore fibrous corona, a key component involved in microtubule capture and spindle assembly checkpoint.