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. 2017 Jul 5;25(7):1145-1152.e4.
doi: 10.1016/j.str.2017.05.016. Epub 2017 Jun 22.

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

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

Angel Rivera-Calzada et al. Structure. .
Free PMC article


The 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.

Keywords: Hsp90 co-chaperone; Pih1; R2TP complex; Rvb1; Rvb2; Tah1; Tel2-Tti1-Tti2; cryo-electron microscopy (cryo-EM).


Figure 1
Figure 1
Assembly and Stoichiometry of R2TP (A) Purification of yeast R2TP in pull-down experiments using a Strep-tag in Tah1p. Fractions from the experiment were analyzed using SDS-PAGE. Fractions where R2TP eluted were selected for EM, in some cases after stabilization using glutaraldehyde. Elution, represents material eluted from the pull-down and used in the negative-stain EM experiments. Beads, represents material that remained bound to the beads after the elution. (B) A gallery of single-molecule images of the R2TP complex, after cross-linking, orientated with the projection of the putative AAA+ ring at the bottom. (C) Representative 2D averages of top and side views of the R2TP complex, after cross-linking, obtained by negative staining. Side view averages show a putative AAA+ ring at the bottom, decorated by extra density on top. (D) Oligomerization state determined by sedimentation velocity in an analytical ultracentrifuge. Sedimentation coefficient distribution c(s) profiles corresponding to purified Tah1p-Pih1p (TP) (dashed line), Rvb1p-Rvb2p (R2) complex (dotted line), and the R2TP complex assembled by mixing TP and R2 complexes (solid line). See also Figure S1.
Figure 2
Figure 2
Cryo-EM of Yeast R2TP and Rvb1p-Rvb2p Hexamers (A) Selected 2D average of a side view of stabilized R2TP obtained by cryo-EM. XL stands for cross-linked. (B) One side view of the low-resolution cryo-EM structure of R2TP stabilized using glutaraldehyde. (C) Selected 2D averages of top and tilted views of R2TP (no cross-linking) obtained by cryo-EM. (D) Two views of the 3D structure of R2 hexamers obtained after classification of the total dataset. α helices corresponding to the C-terminal end of Rvb1p and Rvb2p subunits are identified (circled). DII domains in Rvb1p and Rvb2p have been colored in dark and light pink, respectively. (E) Fitting of an atomic model of Rvb1p-Rvb2p within the EM map. Rvb1p is colored in green and Rvb2p in red. α helices at the C-terminal ends of Rvb1p and Rvb2p subunits are indicated within circles. (F) Comparison between the structure of Rvb1p-Rvb2p hexamers (blue color) and Rvb1p-Rvb2p dodecamers (EMD-3080) (Ewens et al., 2016) (shown as white transparent density). The location of the Rvb2p-DII domain after the putative movement needed to accommodate to its position in Rvb1p-Rvb2p dodecamers is shown in orange color. See also Figure S2.
Figure 3
Figure 3
Structure of Yeast R2TP (A) Several views of the structure of yeast R2TP, with the Rvb1p-Rvb2p AAA+ ring colored in blue, DII domains in pink, and TP in yellow. Due to the differences in resolution, we used a B-factor of −400 for representation of the AAA+ ring, which is solved at higher resolution, whereas the automatic B factor calculated by Relion (−24) was used for the DII-TP. DII domains in Rvb1p and Rvb2p are colored in dark and light pink, respectively. (B) A tilted view of yeast R2TP as in (A), with contacts between TP and DII domains of R2 highlighted (int, internal and ext, external). (C) Schematic of inter-molecular cross-links identified by mass spectrometry. Intra-molecular cross-links and cross-links between Rvb1p and Rvb2p have been omitted for clarity. The cross-link patterns highlight the role of the DII domains of Rvb1p and Rvb2p (pink) and the central segment of Pih1p (yellow) in coupling the TP and R2 sub-complexes. (D) Strep-tag pull-down experiment using Strep-Tah1p-Pih1p complex and a DII-truncated version of Rvb1p and Rvb2p. M indicates Molecular Weight markers. (E) Close-up view of the fitting of Tah1p and Pih1p crystal structures in yeast R2TP. The TP density in the map is shown in transparent density where the crystal structure of the TPR domain of Tah1p (PDB: 4CGU) (red color), the CS domain (PDB: 4CGU), and PIH domain of Pih1p (PDB: 4CGW) (Pal et al., 2014) (yellow color) have been fitted. The relative orientations of the Tah1p-TPR and Pih1p-CS domains have been adjusted from the orientation observed in their co-crystal structure. The positions of the C-terminal MEEVD peptide of Hsp90 bound to Tah1p and the DpSDDE phosphopeptide of Tel2p are indicated (Pal et al., 2014). See also Figures S3 and S4.
Figure 4
Figure 4
Interactions and ATPase Activity in Yeast R2TP (A) Mapping the essential interacting region of Pih1p by pull-down assay. Assembled R2 complex was incubated with various GST fusions to segments of Pih1p. Robust co-precipitation was observed with GST-Pih1p constructs that included residues 215–250 and 230–250 but were eliminated or substantially weakened when residues 215–250 were absent. M indicates Molecular Weight markers. (B) ATPase activity of Rvb1p-Rvb2p is activated by addition of the TP sub-complex. TP was added to R2 (0.5 μM) at the concentrations shown. Values are averages of three replicates and the error bars indicate 1 standard deviation (SD) around the mean value. (C) ATPase activity of Rvb1p-Rvb2p is activated by a Pih1p fragment comprising residues 1–250. Values are averages of three replicates and the error bars indicate 1 SD around the mean value. (D) Cartoon model of the Hsp90-R2TP-TTT super-complex. DII domains are disposed asymmetrically around the Rvb1p-Rvb2p basket and provide the binding sites for one Pih1p-Tah1p complex, which ensures that only a single Hsp90 dimer would be recruited. A unique region between the PIH and CS domains of Pih1p is essential for interaction with R2. The interaction of TP with the flexible DII domains enhances R2 ATPase activity. The Hsp90-binding Tah1p TPR domain and the Tel2p-binding PIH domain of Pih1p face the same side, bringing Tel2p and the Hsp90 chaperone into close proximity.

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