11
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Significance

          Heat shock protein 70 kDa (Hsp70) plays a central role in maintaining protein homeostasis. It cooperates with cochaperone Hsp40, which stimulates Hsp70 ATPase activity and presents protein substrates to Hsp70 to assist refolding. The mechanism by which Hsp40 regulates the intramolecular and intermolecular changes of Hsp70 is still largely unknown. Here, by bulk and single-molecule FRET, we report the conformational dynamics of Hsp70 and its regulation by Hsp40 as well as the kinetics of the multistep Hsp70–Hsp40 functional cycle. We show that Hsp40 modulates the conformations of ATP-bound Hsp70 to a domain-undocked ATPase-stimulated state, and facilitates the formation of a heterotetrameric Hsp70–Hsp40 complex. Our findings provide insights into the functional mechanism of this core chaperone machinery.

          Abstract

          Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

          Related collections

          Most cited references47

          • Record: found
          • Abstract: found
          • Article: not found

          The HSP70 family and cancer.

          The HSP70 family of heat shock proteins consists of molecular chaperones of approximately 70kDa in size that serve critical roles in protein homeostasis. These adenosine triphosphatases unfold misfolded or denatured proteins and can keep these proteins in an unfolded, folding-competent state. They also protect nascently translating proteins, promote the cellular or organellar transport of proteins, reduce proteotoxic protein aggregates and serve general housekeeping roles in maintaining protein homeostasis. The HSP70 family is the most conserved in evolution, and all eukaryotes contain multiple members. Some members of this family serve specific organellar- or tissue-specific functions; however, in many cases, these members can function redundantly. Overall, the HSP70 family of proteins can be thought of as a potent buffering system for cellular stress, either from extrinsic (physiological, viral and environmental) or intrinsic (replicative or oncogenic) stimuli. As such, this family serves a critical survival function in the cell. Not surprisingly, cancer cells rely heavily on this buffering system for survival. The overwhelming majority of human tumors overexpress HSP70 family members, and expression of these proteins is typically a marker for poor prognosis. With the proof of principle that inhibitors of the HSP90 chaperone have emerged as important anticancer agents, intense focus has now been placed on the potential for HSP70 inhibitors to assume a role as a significant chemotherapeutic avenue. In this review, the history, regulation, mechanism of action and role in cancer of the HSP70 family are reviewed. Additionally, the promise of pharmacologically targeting this protein for cancer therapy is addressed.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Human Hsp70 Disaggregase Reverses Parkinson's-Linked α-Synuclein Amyloid Fibrils.

            Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              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.
                Bookmark

                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                7 April 2020
                20 March 2020
                20 March 2020
                : 117
                : 14
                : 7814-7823
                Affiliations
                [1] aNational Laboratory of Biomacromolecules, Chinese Academy of Sciences Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, China;
                [2] bUniversity of the Chinese Academy of Sciences , Beijing 100049, China;
                [3] cZhou Pei-Yuan Center for Applied Mathematics, Tsinghua University , Beijing 100084, China
                Author notes
                4To whom correspondence may be addressed. Email: sarah.perrett@ 123456cantab.net .

                Edited by Lila M. Gierasch, University of Massachusetts at Amherst, Amherst, MA, and approved February 21, 2020 (received for review September 4, 2019)

                Author contributions: S.W. and S.P. designed research; S.W., Y.W., J. Yu, Jie Yang, and Jie Yang performed research; L.H. contributed new reagents/analytic tools; S.W., L.H., Y.W., J. Yu, Jie Yang, Jie Yang, H.Z., and S.P. analyzed data; and S.W., L.H., and S.P. wrote the paper.

                1S.W., L.H., and Y.W. contributed equally to this work.

                2Present address: State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, Guangdong, China.

                3Present address: Department of Cell Biology, Yale School of Medicine, New Haven, CT 06511.

                Author information
                http://orcid.org/0000-0003-0137-0997
                Article
                201914376
                10.1073/pnas.1914376117
                7148561
                32198203
                13829b00-ef76-40cb-a78b-d7454c4cbbe8
                Copyright © 2020 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 10
                Funding
                Funded by: Ministry of Science and Technology of the People's Republic of China (MOST) 501100002855
                Award ID: 2017YFA0504000
                Award Recipient : Sarah Perrett
                Funded by: National Natural Science Foundation of China (NSFC) 501100001809
                Award ID: 31570780
                Award ID: 21673278
                Award ID: 31770829
                Award ID: 21877070
                Award Recipient : Si Wu Award Recipient : Liu Hong Award Recipient : Hong Zhang Award Recipient : Sarah Perrett
                Categories
                Biological Sciences
                Biophysics and Computational Biology

                single-molecule fret,chaperone,hsp70,kinetic analysis,conformational change

                Comments

                Comment on this article