Structural basis for phosphorylation-dependent recruitment of Tel2 to Hsp90 by Pih1

Abstract

Client protein recruitment to the Hsp90 system depends on cochaperones that bind the client and Hsp90 simultaneously and facilitate their interaction. Hsp90 involvement in the assembly of snoRNPs, RNA polymerases, PI3-kinase-like kinases, and chromatin remodeling complexes depends on the TTT (Tel2-Tti1-Tti2), and R2TP complexes-consisting of the AAA-ATPases Rvb1 and Rvb2, Tah1 (Spagh/RPAP3 in metazoa), and Pih1 (Pih1D1 in humans)-that together provide the connection to Hsp90. The biochemistry underlying R2TP function is still poorly understood. Pih1 in particular, at the heart of the complex, has not been described at a structural level, nor have the multiple protein-protein interactions it mediates been characterized. Here we present a structural and biochemical analysis of Hsp90-Tah1-Pih1, Hsp90-Spagh, and Pih1D1-Tel2 complexes that reveal a domain in Pih1D1 specific for binding CK2 phosphorylation sites, and together define the structural basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex.

Graphical abstract

Figure 1

Hsp90-R2TP-Tel2 Connections (A) Schematic of the chain of molecular interactions that connect the Hsp90 chaperone system to the Tel2 component of the TTT complex in yeast. Dotted lines represent interactions structurally defined in this study. N, M, and C represent N-, middle, and C-domains, respectively. TPR, tetratricopeptide repeat domain; CS, CHORD domain-containing protein and Sgt1 domain; and PIH, protein interacting with Hsp90 domain. (B) As in (A), but for the metazoan R2TP system in which the role of Tah1 in yeast is fulfilled by Spagh/RPAP3. Dotted lines represent interactions structurally defined in this study.

Figure 2

Structure of Tah1- Pih1 Complex (A) Secondary structure cartoon of TPR domain of Tah1 (gold) bound to the CS domain of Pih1 (cyan) and a peptide derived from the C terminus of Hsp90 (sticks). (B) Overview of the Tah1-Pih1 complex showing the C-terminal segment of Tah1 (gold, carton and sticks) extending across a shallow hydrophobic depression on the edge of Pih1-CS domain (colored by electrostatic potential: +ve blue → −ve red), and wrapping round to contact the face of the four-stranded β sheet. (C) The proximal part of the Tah1 C terminus segment bridging the edges of the two β sheets of the Pih1-CS domain β sandwich, with a combination of main chain to main chain hydrogen bonds and hydrophobic interactions. (D) Interaction of the distal part of the Tah1 C terminus segment, forming a mixed hydrophobic and polar interface with the face of the four-stranded β sheet. (E) Outside the substantial interaction mediated by the Tah1 C-terminal tail, the juxtaposition of the Tah1-TPR and Pih1-CS domains is fixed by an additional hydrophobic interaction, and an extended polar interface anchored by a bidentate hydrogen bond/ion-pair interaction. (F) Interactions of the Tah1-TPR domain and Hsp90 C-terminal peptide. The Hsp90 peptide binds with compacted conformation stabilized by interaction of the peptide backbone and side chain of Glu 4, with the side chains of Tah1 residues Lys 50, Lys 79, and Arg 83. The α-carboxyl and carboxylate side chain of Asp 7 is bound by a “carboxylate clamp” formed by Tah1 residues Lys 8, Asn 12, Asn 43, and Lys 79. The side chain of Met 3 in the Hsp90 peptide packs against Tyr 82, but is far more exposed than those on other Hsp90-TPR-domain complexes.

Figure 3

TPR Dimerization and Binding to the Hsp90 Dimer (A) SEC of cHsp90, Tah1-Pih1p^187–344, and cHsp90-Tah1-Pih1p^187–344 complex. The elution volume of the cHsp90-Tah1-Pih1p^187–344 complex is consistent with a cHsp90₂-Tah1₂-Pih1p₂^187–344 stoichiometric complex . (B) Exclusive homogenous occupation of both MEEVD sites on the Hsp90 dimer by Tah1. Left-hand gel: α-FLAG immunoprecipitation controls showing immunoprecipitation of FLAG-Tah1, but no interaction with the TPR-domain Hsp90 cochaperone Cpr6 or the dimeric C-terminal domain of Hsp90. A protein band derived from the α-FLAG antibody system is indicated. Right-hand gel: immunoprecipitation of FLAG-Tah1 coprecipitates Hsp90 C-domain, but the yield is diminished in the presence of increasing concentrations of Cpr6. No Cpr6 is precipitated by FLAG-Tah1 even at very high Cpr6 concentrations, showing that mixed loading of Cpr6 and Tah1 onto the Hsp90 C terminus does not occur and that Tah1 exclusively occupies both MEEVD sites simultaneously. (C) SDS-PAGE gel showing migration of native Hsp90 and Tah1 proteins and after crosslinking with DMS (see Experimental Procedures). Tah1 shows a substantial yield of crosslinked dimer comparable in level to that obtained for Hsp90, which is a known obligate dimer.

Figure 4

Structure and Function of Spagh/RPAP3 TPR Domains (A) ITC binding curve for human Hsp90 C-terminal peptide binding to Spagh/RPAP3 at 30°C (left: K_D1 = 1.49 μM and K_D2 = 23.6 μM, two site fitting) and 10°C (right: K_D1 = 1.0 μM and K_D2 = 24 μM, two site fitting). K_D estimates are consistent between the two temperatures indicating an accurate fit to the data. The peptide binds in an ∼2:1 (two Hsp90 peptides to one Spagh/RPAP3) molar ratio consistent with Spagh/RPAP3 mutant binding data (Figures S3A and S3B; TPR1, K_D = 0.94 μM and TPR2, K_D = 9.7 μM). (B) ITC binding curve for Spagh/RPAP3^265–380 binding to full-length Hsp90 dimer with an ∼1:2 molar ratio with K_D = 6.8 μM. (C) Structural comparison of yeast Tah1 TPR-domain (left) and TPR1 (center) and TPR2 domains (right) from Spagh/RPAP3. The N-terminal five helices of the Spagh/RPAP3 TPR domains superimpose with the minimal Tah1-TPR domain. The Cα-trace of the bound Hsp90 C-terminal peptide in each case is shown in magenta. (D) “Carboxyl” clamp interactions with the C terminus of the Hsp90 tail-peptide by Spagh/RPAP3-TPR1 (left) and TPR2 (right). The C-terminal residue of the peptide is anchored on the side chain of Asn 172 in TPR1 and Asn 321 in TPR2. (E) Pull-down of Spagh/RPAP3^120-395 tandem TPR segment by His₆-Hsp90β. Wild-type Spagh/RPAP3 and a TPR1 mutant (N321E) are efficiently coprecipitated, whereas a TPR2 mutant (N172E) diminishes interaction. Mutation of both TPR domains effectively abolishes coprecipitation with Hsp90, confirming the involvement of both TPRs in Hsp90 interaction.

Figure 5

Structure of PIH Domain (A) Secondary structure cartoon of mPih1-PIH domain (left) and yeast Pih1p-PIH domain (right), both rainbow colored. N: blue → C: red. (B) ITC binding curve for peptides from Tel2 spanning the cluster of putative CK2 phosphorylation sites implicated in mediating interaction with Pih1. The unphosphorylated peptide (left) shows no binding, whereas phosphorylation of Tel2-Ser 492 (center), and bis-phosphorylation of Ser 488 and Ser 492 (right) gives submicromolar affinity with K_D = 0.39 and 0.17 μM, respectively. (C) Electrostatic surface (blue: +ve, red: −ve) of mPIH domain cocrystal structure with the bis-phosphorylated Tel2 phosphopeptide. The peptide binds to an intensely basic patch complementary to the multiple negative charges carried by the acidic side chains of the phosphorylated peptide. Only residues 486–496 of the bound peptide were visible. The side chain of pSer 486 was only partially visible. (D) The Tel2-phosphopeptide binds in a compact 3₁₀-helical conformation in a depression formed by the curved face of the central sheet and C-terminal coil segment of the PIH domain. The PIH domain and the mode of binding of the phosphopeptide are quite distinct from previously characterized phosphorylation recognition domains. Only amino acid residues 486–496 of the bound peptide were visible. The side chain of pSer 486 was only partially visible.

Figure 6

PIH Domain–Tel2 Phosphopeptide Interactions (A) Interactions between mPIH domain and Tel2 phosphopeptide. The peptide is well ordered from Leu 490 to Phe 496. The core of the interactions is provided by PIH domain residues Lys 57 and Lys 64, which form a network of charge interactions and hydrogen bonds with the carboxyl side chains of Tel2 residues Asp 491 and Asp 493, and the phosphate moiety of pSer 492. Only residues 486–496 of the bound peptide were visible. The side chain of pSer 486 was only partially visible. (B) Mutation of PIH domain residues Lys 57 (left) or Lys 64 (center) effectively abolishes Tel2-phosphopeptide binding as measured by ITC. Charge reversal of a conserved residue Lys 153, which is not implicated in Tel2 binding by the crystal structure, has no effect on the affinity of the PIH domain for the phosphopeptide (right). (C) Growth of yeast with modified Pih1. Deletion of Pih1 shows a temperature-sensitive (ts) phenotype, whereas deletion of only the CS domain (Pih1^1–163), which mediates recruitment to Hsp90, is not ts. Mutation of residues implicated in phospho-Tel2 binding (K58 equivalent to mouse K64, and K160 equivalent to mouse K166) in isolation did not generate a ts phenotype; however, simultaneous disruption of Tel2 binding and Hsp90 recruitment elicits the ts growth phenotype.

Figure 7

Assembly of the Hsp90-R2TP-TTT Supercomplex (A) Structure of the overall yeast Hsp90-R2TP-TTT supercomplex required for activation of PIKK enzymes such as TOR. The data presented here provide detailed insights into the chain of protein-protein interactions connecting the C terminus of Hsp90 to the TPR domain of Tah1, the C-terminal segment of Tah1 to the CS-domain of Pih1, and the PIH-domain of Pih1 to the CK2 sites on Tel2. Whether Rvb1/2 and Hsp90 directly contact the client PIKK remains to be determined. Tah1 binds Hsp90 as a dimer, occupying both TPR-binding sites on an Hsp90 dimer. However, whether this causes recruitment of a second Pih1-complex and potentially an additional TTT-PIKK complex is unknown. (B) As in (A), but for the metazoan system where Tah1 function is replaced by Spagh/RPAP3, which binds both TPR-binding sites on an Hsp90 dimer as a single polypeptide. The role of the additional N- and C-terminal domains of Spagh/RPAP3 in PIKK activation remains to be determined.