The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System

Abstract

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.

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

“…The N-terminal PIH domain of Pih1p/PIH1D1 binds a CK2-phosphorylation motif on Tel2p/TELO2, mediating recruitment of the TTT complex to R2TP 2 , 3 . Most recently, we have determined the cryo-EM structure of the intact yeast R2TP complex, in which a single Tah1p–Pih1p sub-complex binds a heterohexameric Rvb1–Rvb2 ring 24 , a finding subsequently confirmed by others 25 . In metazoan R2TP, the small (12 kDa) single-TPR domain protein Tah1p is replaced by the much larger (75 kDa) RPAP3/hSpagh whose N-terminal half contains a tandem pair of TPR domains that bind in concert to a single HSP90 dimer 2 .…”

“…The TPR domain of yeast Tah1p mediates interaction with the conserved MEEVD C-terminal tail peptide of HSP90 2 , 19 – 22 , while its C-terminal extension couples Tah1p to the CS-domain of Pih1p 2 , 21 . The central region of Pih1p mediates recruitment of Pih1p–Tah1p to the Rvb1p–Rvb2p heterohexameric ring 23 , 24 . The N-terminal PIH domain of Pih1p/PIH1D1 binds a CK2-phosphorylation motif on Tel2p/TELO2, mediating recruitment of the TTT complex to R2TP 2 , 3 .…”

Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerisation

“…1C). These include seven of the eight members of the Hsp90-R2TP-TTT super-complex (Rivera-Calzada et al, 2017) and one of its target clients, Nipped-A (also known as TRRAP) (Fig. 1C,E).…”

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

“…In various chromatin-remodeling complexes such as INO80 and SRCAP, these ATPases form a scaffold that organizes the architecture of other subunits in the complex (Aramayo et al, 2018;Eustermann et al, 2018;Feng et al, 2018). RUVBL1 and RUVBL2 also interact with RPAP3 and PIH1D1 proteins to form the R2TP complex, a HSP90 co-chaperone involved in the assembly and maturation of some large complexes including RNA polymerase II and members of the Phosphatidylinositol 3-kinase-related kinase (PIKK) family such as ATR, ATM, SMG1 and mTOR (Houry et al, 2018;Martino et al, 2018;Maurizy et al, 2018;Munoz-Hernandez et al, 2019;Rivera-Calzada et al, 2017). In all these complexes, the DIIface of the RUVBL1-RUVBL2 ring is used as scaffold platform for the interaction with other proteins, which are recruited by the DII domains.…”