The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Abstract

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.

Figure 1

TPR2A–TPR2B is the central element for Hsp90 inhibition. (A) Domain architecture of Sti1 and Sti1 fragments. (B) Effects of Sti1 fragments on the yHsp90 ATPase. The ATPase activity of 2 μM yHsp90 was determined using an ATP-regenerative ATPase assay at 30°C. Fragments of Sti1 were tested for inhibition at an equimolar concentration of 2 μM. Data are presented as mean values±s.e. of three independent experiments. (C) Titration of inhibitory fragments (square: Sti1-FL; circle: TPR2A–TPR2B–DP2; triangle: TPR2A–TPR2B). Mean values of three independent experiments are shown. (D) Titration of the Ssa1 peptide PTVEEVD (circles) or the yHsp90 peptide TEMEEVD (squares) to a preformed complex of 2 μM yHsp90 and 2 μM TPR2A–TPR2B.

Figure 2

Structural characterization of the TPR2A–TPR2B module. (A) Ribbon representation of the TPR2A–TPR2B domain. The backbone of the TPR2A–TPR2B fragment is shown in grey. The bound pHsp90 peptides are shown in stick representation in green. The three amino acids (Y390, E421 and R425) involved in the stabilization of the rigid double construct are shown in stick representation in cyan. (B) Zoom of the linker domain as shown in (A). Possible hydrogen bonds are indicated as dashed lines. (C) The TPR2B domain in the presence of the C-terminal Hsp70 heptapeptide (pHsp70). The backbone is shown as ribbon representation in grey and the bound peptide as stick and balls representation in yellow. The electron density of the F_o–F_c map is shown in red at a contour of δ=2. The interacting residues are shown as sticks in grey. Additionally, the residues of the carboxylated clamp, which are not engaged in electrostatic interactions with the peptide, are shown in magenta. (D) Overlay of bound peptides to different TPR domains. Yellow: the pHsp70 heptapeptide bound to Sti1 TPR2B domain (C), green: the pHsp90 pentapeptide bound to the groove of the TPR2A domain in the TPR2A–TPR2B fragment (B), black: the pHsp70 heptapeptide bound to Hop TPR1 domain (pdb entry code: 1ELW) and grey: pHsp90 non-apeptide bound to CHIP (pdb entry code: 2C2L). RMSD for Cα atoms of the last three residues is 0.33, 0.49 and 1.63 for TPR1:pHsp70, TPR2A:pHsp90 and CHIP:pHsp90, respectively. (E, F) Representation of the electrostatic potential modelled onto the accessible molecular surface as calculated and visualized with GRASP and the respective peptide. For clarity, the C-terminal Asp residue of the peptide is referred to as Asp 0 and the preceding residues are numbered in descending order as Val (−1), Glu (−2). (E) Binding of pHsp90 to the TPR2A domain of the TPR2A–TPR2B fragment (same orientation and colour coding as in (A)). (F) Binding of pHsp70 to the TPR2B domain (same orientation and colour coding as in (C)).

Figure 3

Structures of the Sti1 DP1 and DP2 domain. (A) NMR structures of DP1 (left) and DP2 (right) representing the average structure of the 20 lowest-energy structures obtained from simulated annealing calculations after refinement. The additional N-terminal helix of DP1 is coloured in red. (B) Superposition of the 20 lowest-energy structures of DP1 (left) and DP2 (right) obtained from simulated annealing calculations after refinement. (C) Calculated electrostatic potentials for DP1 (left) and DP2 (right). Red indicates negative and blue indicates positive potential.

Figure 4

Binding of TPR2A–TPR2B to the Hsp90-M domain monitored by NMR. (A) (Left) Overlay of the ¹⁵N-TROSY spectra for free TPR2A–TPR2B (black) and in complex with the Hsp90-M domain (red). (Right) Overlay of the ¹⁵N-TROSY spectra for free Hsp90-M (black) and in complex with TPR2A–TPR2B (red). (B) CSP plots for binding of Hsp90-M to ¹⁵N TPR2A–TPR2B (left) and binding of TPR2A–TPR2B to ¹⁵N Hsp90-M (right). Significance levels (average CSP+2x standard deviation) are indicated by a dashed line. (C) Mapping of CSP data from (B) onto the crystal structures of TPR2A–TPR2B (left, pdb: 3uq3) and Hsp90-M (right, pdb: 1hk7). (D) (Left) PRE data for the complex between ¹⁵N-labelled TPR2A–TPR2B and different Proxyl-modified Hsp90-M variants. Signal intensity ratios for the spectra before and after addition of ascorbic acid are plotted against the residue number for each mutant. The domain boundary within the TPR2A–TPR2B fragment is indicated. (Right) Model of the complex between TPR2A–TPR2B and Hsp90-MC obtained by HADDOCK. The unstructured C-terminal ends of Hsp90 not resolved in the crystal structure are drawn in red and the MEEVD motif is represented by red dots.

Figure 5

Influence of Sti1 variants on client activity and complex formation with Hsp90. (A–C) Δsti1 yeast cells expressing Sti1 fragments containing a human GR expression plasmid and a β-galactosidase reporter vector were induced with DOX during exponential growth. Cells were lysed 8–10 h after induction and assayed for β-galactosidase activity and for lysate concentration. Experiments were performed in duplicate with three values each. Error bars indicate standard error. (D) Formation of complexes between Ssa1 and Sti1. Complex formation between Ssa1 and Sti1 variants was analysed by analytical ultracentrifugation. Sedimentation profiles were converted into dc/dt plots according to standard procedures. In all, 0.5 μM of fluorescein-labelled Ssa1 was incubated in the absence of Sti1 fragments (black) or complexed with either 3 μM full-length Sti1 (blue) or TPR2A–TPR2B (green) in 10 mM potassium phosphate, 1 mM TCEP, pH 7.5. Centrifugation was performed at 20°C and 42 000 r.p.m. (E) Formation of ternary complexes between Ssa1 and Sti1–yHsp90. Complex formation between Ssa1 and Sti1 variants was analysed by analytical ultracentrifugation in the presence of access yHsp90. Sedimentation profiles were converted into dc/dt plots according to the standard procedures. In all, 0.5 μM of fluorescein-labelled Ssa1 was incubated in the absence of Sti1 fragments (not shown) or complexed with either 3 μM full-length Sti1 (black), Sti1–N435A (red), TPR2A–TPR2B (green) or TPR2A–TPR2B–N435A (blue) in 10 mM potassium phosphate, 1 mM TCEP, pH 7.5. Centrifugation was performed at 20°C and 42 000 r.p.m.

Figure 6

Model of Sti1 interaction with Hsp90 and Hsp70. Sti1 consists of TPR1 (1–126), DP1 (127–197), TPR2A (257–390), TPR2B (393–520) and DP2 (528–589). The TPR1–DP1 fragment is connected flexibly to the rigid TPR2A–TPR2B block via an unstructured linker region of about 60 residues. The DP2 domain is linked to TPR2B via six unstructured residues. The Hsp90 C-domain binds TPR2A and the Hsp90-M domain TPR2B. This leads to the inhibition of the Hsp90 ATPase. Due to higher affinity, Hsp70 is initially bound to TPR1. DP1 may stabilize the bound client. Subsequently, Hsp70 and client are transferred to TPR2B–DP2. From this position, the client is then transferred to Hsp90.