Krutyholowa, R. | Search bioRxiv

bioRxiv2025

The EF-hand domain of MINDY3 is a Ubiquitin and RAD23 UBL-binding domain

The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire length of the protein except for an atypical EF-hand insertion. We uncover this EF-hand (MINDY3EF-hand) to be a ubiquitin-binding domain with three distinct binding sites, enabling MINDY3 to bind and effectively cleave long polyubiquitin chains. Furthermore, the MINDY3EF-hand domain binds not only to polyubiquitin but also to the UBL domain of the proteasome shuttling and DNA repair factors RAD23A and RAD23B. The MINDY3EF-hand facilitates this interaction with RAD23s in cells and mediates MINDY3 recruitment to DNA damage sites, establishing this unique DUB as a potential regulator of cellular DNA damage responses. MINDY3 binds specifically to the UBL domain of RAD23s, and none of the other UBLs tested. The crystal structure of the MINDY3EF-hand:RAD23AUBL domain complex reveals the molecular basis for specificity. We find that MINDY3 can form a ternary complex with RAD23A/B and polyubiquitin, and our findings suggest a model wherein MINDY3 can deubiquitylate RAD23A/B-bound clients.

biochemistry↗

bioRxiv2023

Closure of the gamma-tubulin ring complex by CDK5RAP2 activates microtubule nucleation

Microtubule nucleation in cells is templated by the {gamma}-tubulin ring complex ({gamma}-TuRC), a 2.3 MDa multiprotein assembly concentrated at microtubule organizing centers (MTOCs). Current {gamma}-TuRC structures exhibit an open conformation that deviates from the geometry of /{beta}-tubulin in the microtubule, potentially explaining their low in vitro microtubule-nucleating activity. Several proteins have been proposed to activate the {gamma}-TuRC, but the mechanisms underlying activation are not known. Here, we isolated the porcine {gamma}-TuRC using CDK5RAP2s centrosomin motif 1 (CM1) and determined its structure with cryo-electron microscopy. 3D heterogeneity analysis revealed an unexpected conformation of the {gamma}-TuRC, in which five protein modules containing MZT2, GCP2, and CDK5RAP2 decorate the outer face of the holocomplex. These decorations drive a long-range constriction of the {gamma}-tubulin ring, bringing the GCP2/GCP3-rich core of the complex in close agreement with the architecture of a microtubule. A purified CDK5RAP2 fragment stimulated the microtubule nucleating-activity of the porcine {gamma}-TuRC as well as a reconstituted, CM1-free human complex in single molecule assays. Our results show that CDK5RAP2 activates the {gamma}-TuRC by promoting {gamma}-tubulin ring closure, providing a structural mechanism for the regulation of microtubule nucleation by CM1 motif proteins in mammals and revealing conformational transitions in {gamma}-tubulin that prime it for templating microtubule nucleation at MTOCs.

biochemistry↗