Munoz Hernandez, H. | bioRxiv Science

bioRxiv2025

CCDC103-mediated assembly of the R2C complex links RUVBL1-RUVBL2 to Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetic disorder caused by defective cilia motility, powered by axonemal dynein motors. Assembly of these motors is facilitated by the molecular chaperone HSP90, its co-chaperone RUVBL1-RUVBL2 and adaptor proteins such as CCDC103 (aka DNAAF19). Mutations in CCDC103 identified in PCD patients impair dynein assembly, contributing to the disease pathology. Here, we present the cryo-electron microscopy structure of the human RUVBL1-RUVBL2-CCDC103 complex at 3.2[A] resolution, a chaperone assembly we refer to as R2C. It comprises a hetero-hexameric RUVBL1-RUVBL2 ring bound to three CCDC103 molecules via their RUVBL2-binding domains (RBDs), which have additional functions. Unlike RPAP3 of R2TP, a previously defined co-chaperone of HSP90, CCDC103 lacks a PIH1D1-binding motif and TPR domains, but its flexible N-terminal region regulates RUVBL1-RUVBL2 oligomerisation. Our characterisation of the R2C complex presented here enhances understanding of the intricate protein network involved in Hsp90-mediated assembly of dynein motors and how adaptor mutations contribute to PCD.

biochemistry↗

bioRxiv2024

Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex

The {gamma}-tubulin ring complex ({gamma}-TuRC) is an essential multiprotein assembly, in which {gamma}-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The {gamma}-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the {gamma}-TuRC. However, the structural basis of the NEDD1-{gamma}-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human {gamma}-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the {gamma}-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric -helical assembly that contacts the lumen of the {gamma}-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down{gamma} -tubulin from cultured cells. The structure of the {gamma}-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the {gamma}-TuRC, but suggest that anchoring of {gamma}-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the {gamma}-TuRC during microtubule nucleation.

biochemistry↗