Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

Structural Genomics Consortium, Karolinska Institutet, Stockholm, Sweden.
PLoS ONE (Impact Factor: 3.73). 01/2010; 5(1):e8625. DOI: 10.1371/journal.pone.0008625
Source: PubMed

ABSTRACT The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.
    PLoS Computational Biology 12/2013; 9(12):e1003379. · 4.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.
    Chemistry & biology 11/2013; · 6.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cross-species transmissions of swine influenza viruses (SIV) raise great public health concerns. Here, subcellular proteomic profiles of human A549 cells inoculated with H3N2 subtype SIV were used to characterize dynamic cellular responses to infection. By two-dimensional gel electrophoresis (2-DE) and mass spectrometry, 27 differentially expressed (13 up-regulated, 14 down-regulated) cytoplasmic proteins and 20 differentially expressed (13 up-regulated, 7 down-regulated) nuclear proteins were identified. Gene Ontology analysis suggested that these differentially expressed proteins were mainly involved in cell death, stress response, lipid metabolism, cell signaling and RNA post-transcriptional modifications. Moreover 25 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between mock- and virus-infected A549 cells. Western blot analysis confirmed that changes in abundance of identified cellular proteins hnRNP U, hnRNP C, ALDH1A1, WARS, IFI35 and HSPB1 in H3N2 SIV-infected cells were consistent with results of 2-DE analysis. By confocal microscopy, nucleus-to-cytoplasm translocation of hnRNP C and colocalization between the viral protein NS1 and hnRNP C as well as NMI were observed upon infection. Ingenuity Pathway Analysis revealed that cellular proteins altered during infection were grouped mainly into NFκB and interferon signaling networks. Collectively, these identified subcellular constituents provide an important framework for understanding host/SIV interactions and underlying mechanisms of SIV cross-species infection and pathogenesis. This article is protected by copyright. All rights reserved.
    Proteomics 09/2013; · 4.43 Impact Factor

Full-text (3 Sources)

Available from
May 30, 2014