Giulia Morra

Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

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.

Methionine Sulfoxides on Prion Protein Helix-3 Switch on the α-Fold Destabilization Required for Conversion

Background The conversion of the cellular prion protein (PrPC) into the infectious form (PrPSc) is the key event in prion induced neurodegenerations. This process is believed to involve a multi-step conformational transition from an α-helical (PrPC) form to a β-sheet-rich (PrPSc) state. In addition to the conformational difference, PrPSc exhibits as covalent signature the sulfoxidation of M213. To investigate whether such modification may play a role in the misfolding process we have studied the impact of methionine oxidation on the dynamics and energetics of the HuPrP(125–229) α-fold. Methodology/Principal Findings Using molecular dynamics simulation, essential dynamics, correlated motions and signal propagation analysis, we have found that substitution of the sulfur atom of M213 by a sulfoxide group impacts on the stability of the native state increasing the flexibility of regions preceding the site of the modification and perturbing the network of stabilizing interactions. Together, these changes favor the population of alternative states which maybe essential in the productive pathway of the pathogenic conversion. These changes are also observed when the sulfoxidation is placed at M206 and at both, M206 and M213. Conclusions/Significance Our results suggest that the sulfoxidation of Helix-3 methionines might be the switch for triggering the initial α-fold destabilization required for the productive pathogenic conversion.

Corresponding Functional Dynamics across the Hsp90 Chaperone Family: Insights from a Multiscale Analysis of MD Simulations

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 ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution.

Molecular switching and ligand-based modulation of the 90-kDa heat-shock protein (Hsp90) chaperone activity may ultimately facilitate conformational coupling to the ATPase cycle along with activation and recruitment of the broad range of client proteins. We present an atomic resolution analysis of the Hsp90 N-terminal domain (NTD) binding energy landscape by simulating protein dynamics with a range of binding partners. We show that the activity of the molecular chaperone may be linked to (i) local folding-unfolding transitions and conformational switching of the “active site lid” upon binding and (ii) differences in the underlying protein dynamics as a function of the binding partner. This study suggests that 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. The present study agrees with the experimental structural data and provides a plausible molecular model for understanding mechanisms of modulation of molecular chaperone activities by binding partners.

Predicting Interaction Sites from the Energetics of Isolated Proteins: A New Approach to Epitope Mapping

An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design.

Molecular mechanism of allosteric communication in Hsp70 revealed by molecular dynamics simulations.

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.

Investigating the mechanism of peptide aggregation: insights from mixed monte carlo-molecular dynamics simulations.

The early stages of peptide aggregation are currently not accessible by experimental techniques at atomic resolution. In this article, we address this problem through the application of a mixed simulation scheme in which a preliminary coarse-grained Monte Carlo analysis of the free-energy landscape is used to identify representative conformations of the aggregates and subsequent all-atom molecular dynamics simulations are used to analyze in detail possible pathways for the stabilization of oligomers. This protocol was applied to systems consisting of multiple copies of the model peptide GNNQQNY, whose detailed structures in the aggregated state have been recently solved in another study. The analysis of the various trajectories provides dynamical and structural insight into the details of aggregation. In particular, the simulations suggest a hierarchical mechanism characterized by the initial formation of stable parallel beta-sheet dimers and identify the formation of the polar zipper motif as a fundamental feature for the stabilization of initial oligomers. Simulation results are consistent with experimentally derived observations and provide an atomically detailed view of the putative initial stages of fibril formation.

Relationship between energy distribution and fold stability: Insights from molecular dynamics simulations of native and mutant proteins.

Most proteins must fold to a well‐defined structure with a minimal stability to perform their function. Here we use a simple, molecular dynamics‐based, energy decomposition approach to map the principal energetic interactions in a set of proteins representative of different folds. This work involves the all‐atom simulation and analysis of the native structures and mutants of five different proteins representative of an all‐alpha (yACPB, Protein A), all‐beta (SH3), and a mixed α/β fold (Proteins G and L). Given a certain structure, a native sequence and a set of mutants, we show that our model discriminates the ability of a mutation to yield a more or less stable protein, in agreement with experimental data, catching the principal energetic determinants of protein stabilization. Our approach identifies the interaction determinants responsible to define a fold and shows that mutations can either modulate the strength of pair‐wise coupling between residues important for folding, or modify the profile of the principal interactions. Furthermore, we address the question of how to evaluate the fitness of a sequence to a given structure by comparing the information contained in the energy map, which recapitulates the chemistry of the sequence, to that contained in the contact map, which recapitulates the fold topology. The results show that the better fit between the energetic properties of the sequence and the fold topology corresponds to a higher stabilization of the protein. We discuss the relevance of these observations to the analysis of protein designability and to the rational evolution of new sequences. Proteins 2008.

Molecular Dynamics Simulations of Proteins and Peptides: From Folding to Drug Design

Computer simulations of proteins, lipids and nucleic acids at equilibrium have become essentially routine. However, the fact remains that complete sampling of conformational space continues to be a bottle-neck in the field. The challenge for the future is to overcome such problems and use computational approaches to understand recognition and spontaneous self-organization in biomolecular systems (folding, aggregation and assembly of complexes), processes that cannot be directly observed experimentally. In this review, examples illustrating the extent to which simulations can be used to understand these phenomena in biomolecular systems will be presented along with examples of methodological developments to increase our physical understanding of the processes. The study cases will cover the problems of peptide-receptor recognition and the use of the information obtained for the design of new non-peptidic ligands; the study of the folding mechanism of small proteins and finally the study of the initial stages of peptide self-aggregation.

Unfolding of the cold shock protein studied with biased molecular dynamics

The cold shock protein from Bacillus caldolyticus is a small β‐barrel protein that folds in a two‐state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native‐like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three‐stranded β‐sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two‐stranded β‐strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them. Proteins 2003;53:000–000.