The structure of the R2T complex reveals a different architecture of the related HSP90 co-chaperones R2T and R2TP

Abstract

Heat shock protein 90 (HSP90) is a molecular chaperone that contributes to the maturation and activation of substrates in multiple cellular pathways. Its activity is supported by various co-chaperones. One of these is R2TP, a complex of RuvBL1-RuvBL2-RPAP3-PIH1D1 in humans, which is involved in the assembly of various multiprotein complexes, including mTORC1 and Box C/D and Box H/ACA snoRNPs. Structural analyses have shown that the complex is organized around a heterohexameric ring of the ATPases RuvBL1-RuvBL2 in both yeast and humans. In addition, several R2TP-like co-chaperones have been identified in humans, such as R2T, which lacks PIH1D1, but these are less well characterized. In seed plants, there are no PIH1D1 orthologs. Here, we have identified the R2T complex of Arabidopsis and determined its cryoEM structure. R2T associates with the prefoldin-like complex in vivo and is located in the cytosolic and nuclear compartments. R2T is organized as a dodecamer of AtRuvBL1-AtRuvBL2a that forms two rings, with one AtRPAP3 anchored to each ring. AtRPAP3 has no effect on the ATPase activity of AtRuvBL1-AtRuvBL2a and binds with a different stoichiometry than that described for human R2TP. We show the interaction of AtRPAP3 with AtRuvBL2a and AtHSP90 in vivo and describe the residues involved. Taken together, our results show that AtRPAP3 recruits AtRuvBL1-AtRuvBL2a and AtHSP90 via a mechanism that is also conserved in other eukaryotes, but that R2T and R2TP co-chaperone complexes have distinct structures that also suggest differences in their functions and mechanisms.