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

Abstract

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.

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

“…For this, RUVBL1 and RUVBL2 were co-expressed and purified as heteromeric complexes containing His-RUVBL1 and RUVBL2 in equimolar amounts, and forming oligomeric complexes where two hexameric rings interact though the DII domains, as revealed by cryo-EM ( Figure 1 -figure supplement 1A), and in agreement with what we described before (Lopez-Perrote et al, 2012;Martino et al, 2018). We first tested the interaction between RUVBL1-RUVBL2 complexes and UPF1 115-914 (a truncated version lacking the flexible N-and C-terminal ends of UPF1), UPF2, UPF3b, EJC and DHX34 by pull-down experiments using the His-tag in RUVBL1 ( Figure 1B, Figure 1 -figure supplement 1B-D).…”

“…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.…”

“…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. One exception is the C-terminal domain of RPAP3 that binds at the ATPase-face of the ring through the interaction with RUVBL2 but not RUVBL1 (Martino et al, 2018;Maurizy et al, 2018;Munoz-Hernandez et al, 2019). ATP binding or hydrolysis by RUVBL1 and/or RUVBL2 is essential to all the reported activities in cells (Izumi et al, 2010;Rajendra et al, 2014;Venteicher et al, 2008), but the purified proteins display very weak ATPase activity in vitro (Nano et al, 2020;Gorynia et al, 2011).…”

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms’ Preassembly

“…RPAP3 ( Figure 2) possesses a C-terminal domain of unknown function (RPAP3-C-ter) and two tandem N-terminal TPRs (tetratricopeptide repeats) domains. The TPR domains enable binding of RPAP3 to Hsp70 [44,45] and Hsp90 (more precisely, to the C-terminal fragment of Hsp90 containing a conserved MEEVD motif/sequence) ( Figure 2) [19,40,46].…”

Sorafenib as an Inhibitor of RUVBL2

“…RUVBL1 and RUVBL2 typically act as a heterohexameric complex, but also function independently [6]. Their sequences can be divided into three domains [7][8][9][10][11][12][13][14][15][16]: (i) an N-terminal αβα subdomain of the AAA+ domain (DI), (ii) an insertion domain unique to the RUVBL proteins (DII) and that is similar in structure to an oligonucleotide/oligosaccharide-binding (OB) fold, which is a ssDNA binding domain present in replication factor protein RPA, and (iii) an all α subdomain of the AAA+ domain (DIII).…”