### abstract ###
Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response.
ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation.
Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function.
Recent studies have illuminated mechanisms related to the chaperone function.
However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle.
Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90.
We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model.
All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer.
We elucidate the mechanisms of signal propagation and determine hot spots involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface.
A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation.
Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface.
Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand.
Our results support a conformational selection model of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.
### introduction ###
Heat Shock Protein 90 is an essential ATPase directed molecular chaperone required for folding quality control, maturation and trafficking of client proteins CITATION CITATION.
Hsp90 represents a fundamental hub in protein interaction networks CITATION, CITATION, with key roles in many cellular functions.
Hsp90 oversees the correct maturation, activation and trafficking among specialized cellular compartments CITATION of a wide range of client proteins CITATION, CITATION, CITATION.
The functions of clients range from signal transduction to regulatory mechanisms and immune response CITATION.
Client proteins typically include numerous kinases, transcription factors and other proteins that serve as nodal points in integrating cellular responses to multiple signals CITATION.
Given its role at the intersection of fundamental cellular pathways, it is becoming increasingly clear that Hsp90 deregulation can be associated with many pathologies ranging from cancer to protein folding disorders and neurological diseases CITATION, CITATION.
Because of this role in disease development, pharmacological suppression of Hsp90 activity has become an area of very intense research, in molecular oncology in particular.
Targeted suppression of Hsp90 ATPase activity with a small molecule inhibitor, the benzoquinone ansamycin antibiotic 17-allylamino-17-demethoxygeldanamycin, and some of its derivatives CITATION, CITATION, has shown promising anticancer activity in preclinical models and has recently completed safety evaluation in humans CITATION.
Further clinical trials have also been initiated with other small molecules also used in drug combinations in various cancer types CITATION .
Hsp90 operates as a dimer in a complex cycle driven by ATP binding and hydrolysis and by ATP/ADP exchange.
Initial structural efforts concentrated on isolated, individual domains of human CITATION CITATION or yeast Hsp90 CITATION, CITATION, CITATION CITATION, the ER homologue Grp94 CITATION, CITATION or the Escherichia coli homologue, HtpG CITATION, CITATION.
The crystal structures of larger constructs have also been reported CITATION, CITATION.
The first X-ray crystal structures of full-length Hsp90 from yeast bound to the ATP mimic AMPPNP revealed a homodimeric structure in which the individual protomers have a twisted parallel arrangement CITATION.
Each protomer, in turn, is characterized by a modular architecture with three well-defined domains: an N-terminal regulatory Domain, responsible for ATP binding, a Middle Domain, which completes the ATPase site necessary for ATP hydrolysis and binds client proteins, and a C-terminal dimerization Domain which is required for dimerization CITATION.
The same global topology is shared by the ATP-bound states of the E.coli homolog HtpG CITATION and by the Endoplasmatic Reticulum paralog Grp94 CITATION.
Interestingly, crystal structures of the full-length constructs for Htpg or Grp94 in complex with either ADP or in the apo state showed substantially different conformations.
The HtpG apo state adopted an open structure in which each of the three domains exposed hydrophobic surface area, while in the ADP-bound form these hydrophobic surfaces clustered to form a more compact state CITATION .
Structural and biochemical studies of the solution state of Hsp90 and its complexes using small angle X-ray scattering CITATION have provided the first experimental evidence of a highly dynamic and stochastic nature of Hsp90, whereby the equilibrium between different conformational states of the molecular chaperone can be readily shifted to recruit a Hsp90 conformation that is suitable for efficient Cdc37 co-chaperone recognition.
More recent solution structure data obtained using SAXS, single particle cryo-electron microscopy and modeling approaches showed that the apo-Hsp90 dimer may be in equilibrium among different open, extended states, still preserving the constitutive dimerization provided by the CTDs, and that nucleotide binding may shift this equilibrium towards compact conformations CITATION CITATION.
In particular, SAXS data have revealed that the ADP-bound compact state of HtpG can be in equilibrium with an extended state, which could be significantly populated in the absence of crystal packing effects CITATION.
In contrast, crystal structures of AMPPNP and ADP-bound forms of the ER-paralog Grp94 showed that there is relatively little difference in conformation between the two nucleotide bound states in the crystal CITATION, representing extended structures.
Recent studies based on mutation analysis, cross-linking and electron microscopy CITATION, CITATION suggested that different, compact states can be accessed by Grp94 in the presence of ATP.
These studies have indicated that upon binding to a specific partner, functional states of Hsp90 can be recruited using the intrinsic conformational flexibility of Hsp90.
Although the exact mechanism of coupling between ATP-binding/hydrolysis and client protein folding is still unclear, the combination of X-ray structural observations and biochemical data supports a picture in which the chaperone undergoes conformational rearrangements bringing the two NTDs in close association in the ATP-bound state, but not in the ADP-bound or apo states.
This defines a conformational cycle that involves constitutive dimerization through the CTDs and transient, ATP-dependent dimerization of the NTDs in a molecular clamp mechanism.
In terms of intrinsic protein dynamics, the mechanism of conformational coupling to the ATPase cycle involves a tense, structurally rigid conformational state of Hsp90 upon ATP binding, whereas a subsequent hydrolysis to ADP leads to a more relaxed, structurally flexible state of Hsp90 CITATION, CITATION, CITATION.
Finally, in the nucleotide-free form, the dimer moves to an open state.
The crystal structures of the full-length dimer also highlight the remarkable flexibility of the ATP-lid, a segment composed of two helices and the intervening loop located immediately adjacent to the ATP binding site CITATION.
The lid is displaced from its position in the isolated Hsp90 NTD structure and folds over the nucleotide pocket to interact with the bound ATP yielding the closed conformation indicating its possible importance in the progression of the chaperone cycle.
These studies are reminiscent of the results from an H/D exchange mass spectrometry investigations on the human Hsp90 in solution, which showed that the co-chaperone and inhibitor binding to the NTD can induce conformational changes at the Hsp90 domain-domain interfaces CITATION.
Moreover, Frey and coworkers CITATION have shown that kinetic and equilibrium binding constants depend on the intrinsic conformational equilibrium of the Hsp90 obtained from different species, reflecting differential affinity and reactivity towards ATP.
The kinetic analysis of the ATPase cycle has suggested that during the ATPase cycle Grp94 may be predominantly in the open state.
In contrast in the yeast Hsp90 the open state is only populated to 20 percent and a closed structure is observed in the presence of nucleotides CITATION.
Hence, conformational transitions during the ATPase cycle are structurally similar for different Hsp90 proteins, while the energetic balance between individual steps may be species-dependent, which is manifested in differences in the binding kinetics CITATION.
Overall, the solution data have suggested that the molecular mechanism of the Hsp90 chaperone cycle can be more adequately described as a stochastic process, in which ATP binding can shift the intrinsic conformational equilibrium of Hsp90 between the open apo state, the ADP-bound compact and the ATP-bound, closed protein state seen in different crystal structures.
The most recent structural studies of the apo and nucleotide-bound conformations of the E. coli, yeast, and human Hsp90 homologs have further supported the existence of a universal three-state conformational cycle for Hsp90, consisting of open-apo, ATP-closed and ADP-compact nucleotide-stabilized states, whereby the intrinsic conformational equilibrium between these states can be highly-species dependent CITATION.
According to these results, the evolutionary pressure may act through thermodynamic stabilization of the functionally relevant Hsp90 conformations recruited from the conformational equilibrium, to ensure the adequate response to the presence of organism-specific co-chaperones and protein clients.
Importantly, ATP or ADP binding can shift the conformational equilibrium far away from the apo state for E. coli and yeast Hsp90, whereas the conformational equilibrium for human Hsp90 is largely dominated by the open form, even in the presence of the nucleotide binding.
Strikingly, this study has shown that nucleotide binding provides only small stabilization energy, thereby biasing, rather than determining, the occupancy of different conformational states existing in a dynamic equilibrium.
Overall, the intrinsic conformational flexibility of Hsp90 is critical to the molecular chaperon cycle, including structural adaptation to diversity of co-chaperones and client proteins CITATION.
Different steps in the cycle are accompanied by binding to different co-chaperone proteins with specific functions.
The Hop co-chaperone, for instance, arrests ATP hydrolysis and binds simultaneously to the Hsp70 molecular chaperone, coupling the two systems.
The Hop binding to Hsp90 involves interactions at both M-domains and CTD domains CITATION, CITATION stabilizing a conformation that is incompetent for ATP hydrolysis and N-terminal dimerization CITATION.
In contrast, the stress-regulated co-chaperone Aha1 substantially increases ATPase rates increasing Hsp90 chaperone activities CITATION.
In the case of binding to other co-chaperones, Cpr6 and Sba1, it was shown that ATP-binding and hydrolysis is required to ensure productive complex formation: interestingly, Sba1 binds to the NTD while Cpr6 binds to the CTD CITATION.
These observations suggest a role for the nucleotide in selecting and stabilizing different conformations of Hsp90, related to specific different functions in the chaperone cycle CITATION .
These crystallographic, cryo-EM, SAXS and three-dimensional single-particle reconstruction studies, applied to the isolated Hsp90 domains and full Hsp90 dimer in different species, have provided a wealth of novel insights into the molecular mechanism and function of Hsp90.
However, there are still a number of important unresolved problems concerning the atomic resolution understanding of the interplay between ligand binding and the global functional motions of the molecular chaperone.
We have recently performed computational studies of the Hsp90 conformational dynamics and analyzed at atomic resolution the effects of ligand binding on the energy landscape of the Hsp90 NTD by all-atom MD simulations.
MD simulations of Hsp90 NTD have been carried out for the apo protein and Hsp90 complexes with its natural ligands ATP, ADP, small molecule inhibitors, and peptides CITATION.
These simulations have clarified the role of ATP-lid dynamics, differences in local conformational changes and global flexibility, as well as the functional interplay between protein rigidity and entropy of collective motions depending on the interacting binding partners.
We have found that the energy landscape of the apo Hsp90 NTD may be populated by structurally different conformational states, featuring local conformational switching of the ATP-lid which is accessible on longer time scales.
The results of this study have suggested a plausible molecular model for understanding the mechanisms of modulation of molecular chaperone activities by binding partners.
According to this model, structural plasticity of the Hsp90 NTD can be exploited by the molecular chaperone machinery to modulate enhanced structural rigidity during ATP binding and increased protein flexibility as a consequence of the inhibitor binding.
CITATION .
The molecular basis of signal propagation mechanisms and inter-domain communication pathways in the Hsp90 as a function of binding ligands cannot be inferred directly from crystallographic studies.
As a result, computational approaches are instrumental in revealing the atomic details of inter-domain communication pathways between the nucleotide binding site and distant CTD, which may be involved in governing the chaperone equilibrium between major conformational states.
In this work, we have embarked on a comprehensive computational analysis of Hsp90 dynamics and binding which provides important insights into our understanding of the Hsp90 molecular mechanisms and function at atomic resolution.
We describe large-scale MD simulations to study the conformational motions and inter-domain communication pathways of the full-length yeast Hsp90 in three different complexes: with ATP, with ADP and in the apo form.
In support of the experimental hypotheses, our results provide atomic models of a cross-talk between N- and C-terminal binding sites that may induce an allosteric regulation of the complex molecular chaperone machinery.
These results of our study suggest that the low-resolution features of communication pathways in the Hsp90 complexes may be determined by the inherent topological architecture of the chaperone, yet specific signal communication pathways are likely to be selected and activated based on the nature of the binding partner.
