Extended conformational states dominate the Hsp90 chaperone dynamics

Abstract

The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.

Keywords: MD simulations; NMR; chaperone; coarse-grained simulation; heat shock protein (HSP); heat shock protein 90 (Hsp90); molecular dynamics; small-angle X-ray scattering.

Figure 1

Overview of the Hsp90 dimer conformations. The closed X-ray structure of the Hsp90 dimer (PDB ID: 2CG9) with a modeled charged linker (CL) region (NTD in blue, CL in red, MD in green, CTD in yellow) (top, left). The rich linker dynamics obtained from aMD simulations is illustrated on the left side of the Hsp90 dimer. The partially open X-ray structure of GRP94 (PDB ID: 2O1U, top right), a homologous to Hsp90, and open dimer (PDB ID: 2IOQ, bottom left). The pair-distance distribution of the crystal structures is compared to profiles obtained from SAXS experiments (bottom right). Key features of the experimental P(r) profile are not reproduced by the X-ray structures. CTD, C-terminal domain; MD, middle domain; NTD, N-terminal domain; PDB, Protein Data Bank.

Figure 2

Conformational dynamics of the NTD-MD construct of Hsp90.A, distribution of the radius of gyration (R_g) obtained from SAXS reweighted aMD ensembles of the NTD-MD and NTD-MD_ΔCL constructs. The extended, partially open, and compact conformations are defined based on the number of residue contacts between the NTD (residue 20–202) and MD (residue 290–524) (0 for extended, >10 for the compact state). The intercept shows the ratio between compact, partially open, and extended states. B, structure of a highly extended conformation. C, pair-distance distribution of the reweighted NTD-MD ensemble. D, free energy profile of the compact state shown based on the torsion angles defined by the center of mass of residues 47 to 60, 178 to 188, 385 to 392, and 404 to 409 (torsion angle 1, in orange) and residues 64 to 69, 28 to 34, 395 to 403, and 367 to 370 (torsion angle 2, in gray). E, representative structures of highlighted regions in (D). aMD, atomistic molecular dynamics; MD, middle domain; NTD, N-terminal domain; SAXS, small-angle X-ray scattering.

Figure 3

Dynamics of the full-length Hsp90.A and B, radius of gyration (R_g) as a function of maximum intermolecular distance (D_max) of the full-length. A, Hsp90 and (B) Hsp90_ΔCL ensemble. The size of the circles represents the weight of the individual conformations, while the color corresponds to the opening angle between MDs and the center of the CTDs. C, ratio between extended (no contacts between NTDs [residue 20–202] and MDs [residue 290–524]) and compact (contacts between NTDs and MDs of both chains) states. D, representative snapshot of the open “V-shape” conformation sampled during cgMD simulations. The representation shows the backbone beads and elastic network and bonds (NTD in blue, CL in red, MD in green, CTD in yellow). cgMD, coarse-grained molecular dynamics; CL, charged linker; CTD, C-terminal domain; MD, middle domain; NTD, N-terminal domain.

Figure 4

Role of the extended conformational landscape of Hsp90. In the absence of clients and cochaperones, Hsp90 exists in an equilibrium of open conformations involving detachment of the N-terminal domain (NTD) from the middle domain (MD, panels 1 and 2). These extended conformations allow the rotation and docking of the NTD with the MD upon nucleotide binding (panel 3), in a relative arrangement similar to that of the closed conformation of the dimer (panel 4). The binding of clients and cochaperones to specific regions of Hsp90 cause the shift toward the fully closed, active conformation of the dimer (panel 4).