RESUMEN
Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Centrosoma , Microtúbulos , Humanos , Centrosoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Microtúbulos/metabolismo , Centriolos/metabolismo , Centriolos/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Mutación , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Proteínas NuclearesRESUMEN
OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive. Here, we use a battery of biophysical, biochemical, and structural analyses to elucidate the molecular basis of the OLA1-BARD1 interaction. Our structural and enzyme kinetics analyses show this nucleotide-dependent interaction enhances the ATPase activity of OLA1 by increasing the turnover number (kcat). Unlike canonical GTPase activating proteins that act directly on the catalytic G domain, the BARD1 BRCT domain binds to the OLA1 TGS domain via a highly conserved BUDR motif. A cancer related mutation V695L on BARD1 is known to associate with centrosome abnormality. The V695L mutation reduces the BARD1 BRCT-mediated activation of OLA1. Crystallographic snapshot of the BRCT V695L mutant at 1.88 Å reveals this mutation perturbs the OLA1 binding site, resulting in reduced interaction. Altogether, our findings suggest the BARD1 BRCT domain serves as an ATPase activating protein to control OLA1 allosterically.