The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1–RUVBL2–RPAP3–PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.

Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.

The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2–containing complexes.

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.

SummaryThe R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldinlike (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.

Highlights d TELO2-TTI1-TTI2 (TTT) forms a direct complex with RUVBL1-RUVBL2 (R2) d TTT interacts with two consecutive DII domains in the heterohexameric R2 ring d TTT inhibits R2 ATPase and antagonizes RPAP3/Tah1p-PIH1D1/Pih1p engagement d TTI1-TTI2 binds the kinase region of mTOR but does not inhibit its catalytic activity

Highlights d TELO2-TTI1-TTI2 (TTT) forms a direct complex with RUVBL1-RUVBL2 (R2) d TTT interacts with two consecutive DII domains in the heterohexameric R2 ring d TTT inhibits R2 ATPase and antagonizes RPAP3/Tah1p-PIH1D1/Pih1p engagement d TTI1-TTI2 binds the kinase region of mTOR but does not inhibit its catalytic activity