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. Dynamic processes underlie the functions of all proteins. Hence, to understand, control, and design protein functions in the cell, we need to unravel the basic principles of protein dynamics. This is fundamental in studying the mechanisms of a specific class of proteins known as molecular chaperones, which oversee the correct conformational maturation of other proteins. In particular, molecular chaperones of the stress response machinery have become the focus of intense research, because their upregulation is responsible for the ability of tumor cells to cope with unfavorable environments. This is largely centered on the expression and function of the molecular chaperone Hsp90, which has provided an attractive target for therapeutic intervention in cancer. Experiments have shown that the chaperone functions through a nucleotide-directed conformational cycle. Here, we show that it is possible to identify the effects of nucleotide-related chemical differences on functionally relevant motions at the atomic level of resolution. The protein may fluctuate at equilibrium among different available dynamic states, and binding of a specific partner may shift the equilibrium toward the thermodynamically most stable complexes. These results provide us with important mechanistic insight for the identification of new regulatory sites and the design of possible new drugs.

Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones. Understanding the connections between structure, binding, dynamics and function in proteins is one of the most fascinating problems in biology and is actively investigated experimentally and computationally. In the latter context, significant advancements are possible by exposing the causal link between the fine atomic-scale protein-ligand interactions and the large-scale protein motions. One ideal avenue to explore this relationship is given by proteins of the Hsp90 chaperones family. Their dynamics is regulated by ATP binding and hydrolysis, which activates the onset of large-scale, functional conformational changes. Herein, we concentrated on three homologs with markedly different structural organization—mammalian Grp94, yeast Hsp90 and prokaryotic HtpG—and developed a novel computational multiscale approach to detect and characterize the salient traits of the functionally-oriented internal dynamics of the three chaperones. The comparative analysis, which exploits a novel highly simplified, yet viable, description of the protein internal dynamics, highlights fundamental mechanical aspects that preside the ligand-dependent conformational arrangements in all chaperones. For the three molecules, two corresponding regions are singled out as ligand-susceptible hinges for the large-scale internal motion. On the basis of this and other evidence it is suggested that these regions represent functionally relevant druggable substructures in the discovery of novel allosteric modulators.