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      Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations


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          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.

          Author Summary

          Allostery, or the capability of proteins to respond to ligand binding events with a variation in structure or dynamics at a distant site, is a common feature for biomolecular function and regulation in a large number of proteins. Intra-protein connections and inter-residue coordinations underlie allosteric mechanisms and react to binding primarily through a finely tuned modulation of motions and structures at the microscopic scale. Hence, all-atom molecular dynamics simulations are suitable to investigate the molecular basis of allostery. Moreover, understanding intra-protein communication pathways at atomistic resolutions offers unique opportunities in rational drug design. Proteins of the Hsp70 family are allosteric molecular chaperones involved in maintaining cellular protein homeostasis. These proteins are involved in several types of cancer, neurodegenerative diseases, aging and infections and are therefore pharmaceutically relevant targets. In this work we have analyzed, by multiple molecular dynamics simulations, the long-range dynamical and conformational effects of ligands bound to Hsp70, and found relevant differences in comparison to the known non-allosteric structural homolog Hsp110. The resulting model of the mechanism of allosteric propagation offers the opportunity of identifying on-pathway allosteric druggable sites, which we propose could guide rational drug-design efforts targeting Hsp70.

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          Most cited references42

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          Hsp70 chaperones: Cellular functions and molecular mechanism

          Abstract. Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.
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            Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.

            DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of +/-35 degrees with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.
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              Routine Access to Millisecond Time Scale Events with Accelerated Molecular Dynamics

              In this work, we critically assess the ability of the all-atom enhanced sampling method accelerated molecular dynamics (aMD) to investigate conformational changes in proteins that typically occur on the millisecond time scale. We combine aMD with the inherent power of graphics processor units (GPUs) and apply the implementation to the bovine pancreatic trypsin inhibitor (BPTI). A 500 ns aMD simulation is compared to a previous millisecond unbiased brute force MD simulation carried out on BPTI, showing that the same conformational space is sampled by both approaches. To our knowledge, this represents the first implementation of aMD on GPUs and also the longest aMD simulation of a biomolecule run to date. Our implementation is available to the community in the latest release of the Amber software suite (v12), providing routine access to millisecond events sampled from dynamics simulations using off the shelf hardware.

                Author and article information

                Role: Editor
                PLoS Comput Biol
                PLoS Comput. Biol
                PLoS Computational Biology
                Public Library of Science (San Francisco, USA )
                December 2012
                December 2012
                27 December 2012
                : 8
                : 12
                [1 ]Istituto di Tecnologie Biomediche – Consiglio Nazionale delle Ricerche (ITB-CNR), Segrate (Mi), Italy
                [2 ]Istituto di Chimica del Riconoscimento Molecolare - Consiglio Nazionale delle Ricerche (ICRM-CNR), Milano, Italy
                University of Wisconsin, Madison, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GC GM FC IM. Performed the experiments: FC GM IM. Analyzed the data: FC GM. Contributed reagents/materials/analysis tools: IM GC LM. Wrote the paper: FC GM GC. Coordinated the research: GC LM Critically reviewed the manuscript: IM LM.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 15
                This work has been supported by the Italian Ministry of Education and Research through the Flagship (PB05) “InterOmics”, ITALBIONET (RBPR05ZK2Z), HIRMA (RBAP11YS7K) and the European “MIMOMICS” projects. GC acknowledges support from AIRC (Italian Association for Cancer Research) project IG.11775 and from Fondazione Cariplo through the CHECOSP project 2011.1800. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Computational Biology
                Chemical Biology

                Quantitative & Systems biology
                Quantitative & Systems biology


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