Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3TA, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2011; 108(44):17939-44. DOI: 10.1073/pnas.1106261108
Source: PubMed


The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K(D) values. This simultaneous calculation of the K(D)s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K(D) values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle.

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    • "Because detergent removal is dictated by the physical properties of the detergent micelle (Reading et al., 2015), it is not possible to compare accelerating voltage conditions for constructs in different detergents. The relative abundances of oligomers were calculated using the UniDec deconvolution software program (Marty et al., 2015) with detector efficiency taken into account (Ebong et al., 2011; Fraser, 2002; Stengel et al., 2012). "
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    ABSTRACT: The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 05/2015; 22(5):593-603. DOI:10.1016/j.chembiol.2015.04.016 · 6.65 Impact Factor
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    • "In previous studies, we established that Hsp70 was essentially monomeric under our solution conditions (Ebong et al., 2011), although dimerization has been reported previously in solution and X-ray structures of DnaK (Qi et al., 2013). Recently, specific mutations of DnaK were designed to disrupt the dimer interface observed crystallographically and to probe its functional significance (Sarbeng et al., 2015). "
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    ABSTRACT: Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 04/2015; 11(5). DOI:10.1016/j.celrep.2015.03.063 · 8.36 Impact Factor
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    • "However, biochemical studies indicate that one Hop monomer is sufficient to inhibit the ATPase activity of the Hsp90 dimer [48], and ultracentrifugation confirmed this 2:1 stoichiometry under physiologically relevant conditions [30]. Thus, binding of the first Hop molecule to Hsp90 appears to reduce the affinity for the second [47]. These observations indicate that Hop binding to Hsp90 induces asymmetry. "
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    ABSTRACT: Hsp90 is a molecular chaperone that facilitates the maturation of signaling proteins including many kinases and steroid hormone receptors. Through these client proteins, Hsp90 is a key mediator of many physiological processes and has emerged as a promising drug target in cancer. Additionally, Hsp90 can mask or potentiate the impact of mutations in clients with remarkable influence on evolutionary adaptations. The influential roles of Hsp90 in biology and disease have stimulated extensive research into the molecular mechanism of this chaperone. These studies have shown that Hsp90 is a homodimeric protein that requires ATP hydrolysis and a host of accessory proteins termed co-chaperones to facilitate the maturation of clients to their active states. Flexible hinge regions between its three structured domains enable Hsp90 to sample dramatically distinct conformations that are influenced by nucleotide, client and co-chaperone binding. While it is clear Hsp90 can exist in symmetrical conformations, recent studies have indicated that this homodimeric chaperone can also assume a variety of asymmetric conformations and complexes that are important for client maturation. The visualization of Hsp90-client complexes at high resolution together with tools to independently manipulate each subunit in the Hsp90 dimer are providing new insights into the asymmetric function of each subunit during client maturation. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 04/2015; 427(18). DOI:10.1016/j.jmb.2015.03.017 · 4.33 Impact Factor
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