Molecular dynamics simulation techniques have been used to investigate the effect of 2,2,2-trifluoroethanol (TFE) as a cosolvent on the stability of three different secondary structure-forming peptides: the ␣-helix from Melittin, the three-stranded ␤-sheet peptide Betanova, and the ␤-hairpin 41-56 from the B1 domain of protein G. The peptides were studied in pure water and 30% (vol͞vol) TFE͞water mixtures at 300 K. The simulations suggest that the stabilizing effect of TFE is induced by the preferential aggregation of TFE molecules around the peptides. This coating displaces water, thereby removing alternative hydrogen-bonding partners and providing a low dielectric environment that favors the formation of intrapeptide hydrogen bonds. Because TFE interacts only weakly with nonpolar residues, hydrophobic interactions within the peptides are not disrupted. As a consequence, TFE promotes stability rather than inducing denaturation. F or more than three decades, 2,2,2-trifluoroethanol (TFE) has been used as a cosolvent for the study of peptides in solution because NMR and CD studies show that the presence of TFE increases the population of ␣-helix and ␤-sheet content in secondary-structure-forming peptides in TFE͞water mixtures (1-3). Despite the effects of TFE having been known for such a long time, the mechanism by which TFE stabilizes secondary structure in peptides is still not clear. One possible explanation is the preferential solvation of the folded state by TFE (3). According to this hypothesis, TFE acts within the context of a preexisting helix-coil equilibrium, and the preferential interaction of TFE with the folded state shifts the equilibrium toward more structured conformations (3). The molecular nature of the TFE-peptide interaction is not clear, however. Alternative mechanisms have also been proposed to explain the stabilizing effect of TFE. In particular, the effect could result from TFE reinforcing hydrogen bonds between carbonyl and amidic NH groups by the removal of water molecules in the proximity of the solute (4) and͞or the lowering of the dielectric constant (1). Furthermore, small-angle x-ray-scattering studies (2) show that TFE forms clusters in water as the concentration of the organic cosolvent is increased, with a maximum at 30% (vol͞vol) TFE. Reiersen and Rees (5) have proposed that such TFE clusters locally assist the folding of secondary-structure elements by providing a solvent matrix that promotes hydrophobic interactions between amino acid side chains. Recent NMR studies involving small peptides in TFE provide some support for this hypothesis (6-8). Each of these mechanisms could explain the stabilization of ␣-helical peptides in solution (1, 3) but not necessarily the stabilization of ␤-structure (1, 2).In recent years molecular dynamics (MD) simulations have been increasingly used to understand the complex conformational equilibria of polypeptides in solution and to predict structural preferences (9-11). In particular, the importance of side-chain interactions in determining pepti...

Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents. Hsp90 is a chaperone protein that controls the folding of proteins in multiple signaling networks that drive tumor development and progression. Here, we report the synthesis and properties of Gamitrinibs, a class of small molecules designed to selectively target Hsp90 in human tumor mitochondria. Gamitrinibs were shown to accumulate in the mitochondria of human tumor cell lines and to inhibit Hsp90 activity by acting as ATPase antagonists. Unlike Hsp90 antagonists not targeted to mitochondria, Gamitrinibs exhibited a "mitochondriotoxic" mechanism of action, causing rapid tumor cell death and inhibiting the growth of xenografted human tumor cell lines in mice. Importantly, Gamitrinibs were not toxic to normal cells or tissues and did not affect Hsp90 homeostasis in cellular compartments other than mitochondria. Therefore, combinatorial drug design, whereby inhibitors of signaling networks are targeted to specific subcellular compartments, may generate effective anticancer drugs with novel mechanisms of action.

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.

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.

Summary Swe1 (Saccharomyces WEE1), the only “true” tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1Wee1 phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90α) in the N-domain of Hsp90. Phosphorylation is cell cycle-associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Non-phosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific post-translational modification of a unique subcellular pool of the chaperone, and they provide a novel strategy to increase the cellular potency of Hsp90 inhibitors.

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.

The study of allosteric functional modulation in dynamic proteins is attracting increasing attention. In particular, the discovery of new allosteric sites may generate novel opportunities and strategies for drug development, overcoming the limits of classical active-site oriented drug design. In this paper, we report on the results of a novel, ab initio, fully computational approach for the discovery of allosteric inhibitors based on the physical characterization of signal propagation mechanisms in proteins and apply it to the important molecular chaperone Hsp90. We first characterize the allosteric "hot spots" involved in interdomain communication pathways from the nucleotide-binding site in the N-domain to the distal C-domain. On this basis, we develop dynamic pharmacophore models to screen drug libraries in the search for small molecules with the functional and conformational properties necessary to bind these "hot spot" allosteric sites. Experimental tests show that the selected moelcules bind the Hsp90 C-domain, exhibit antiproliferative activity in different tumor cell lines, while not affecting proliferation of normal human cells, destabilize Hsp90 client proteins, and disrupt association with several cochaperones known to bind the N- and M-domains of Hsp90. These results prove that the hits alter Hsp90 function by affecting its conformational dynamics and recognition properties through an allosteric mechanism. These findings provide us with new insights on the discovery and development of new allosteric inhibitors that are active on important cellular pathways through computational biology. Though based on the specific case of Hsp90, our approach is general and can readily be extended to other target proteins and pathways.

In this paper we describe the first all-atom aqueous-phase MD simulations of human carbonic anhydrase II in three protonation states relevant to the rate-limiting intramolecular proton-transfer step. In particular, we have examined the zinc−water form of the enzyme (CHOH), the zinc−hydroxide form of the enzyme with a doubly protonated His-64 (COHH, the putative intramolecular proton-transfer proton-accepting residue), and the native zinc−hydroxide form (COH) of the enzyme (i.e., with an unprotonated His-64). From these MD simulations (up to ∼1 ns in length) we have studied in detail the dynamics of these three systems. Overall the dynamics of the three systems do not vary significantly (e.g., the active site region is rigid, the number of long-lived hydrogen bonds is constant, etc.) with the exception of COHH. In this case the residues that line the entrance to the active site cavity (near the location of His-64) undergo significantly higher fluctuations than in the CHOH and COH cases. It is postulated that this facilitates solvent and buffer exchange around His-64, thereby facilitating the intermolecular proton-transfer step. We also find that the motion of His-64 is limited in all three cases to occupying the “in” orientation (∼7 Å from the zinc ion, while the so-called “out” conformer is further away), which suggests that fluctuations of this residue between the in and out conformers have a limited influence on the intramolecular proton transfer. However, due to the limited time scales of our simulations, this needs to be examined in more detail. Importantly, though, we find that His-64 acts as a “gate-keeper” between the inner active site region (characterized by localized water molecules) and the outer (bulk) region, which is characterized by relatively freely diffusing water molecules. This function of His-64 has not been realized previously. In the inner active site we have identified relatively long-lived water bridges between the zinc-bound water or hydroxide and the imidazole or imidazolium side chain of His-64. The lengths of these bridges vary between two and six water molecules, and the preferred bridge depends on the protonation of the active site. We estimate that the probability of water bridge formation is low (at most ∼1.5 kcal/mol) and that water bridge formation is not the rate-limiting step in the proton-transfer process (transfer from zinc-bound water to an active site water is rate-limiting).