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Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB
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Abstract
Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s −1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.
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Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764
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Research works printed but not linked: 56
- Avellaneda, 2020, Processive extrusion of polypeptide loops by a Hsp100 disaggregase, Nature, 578, 317, 10.1038/s41586-020-1964-y · DOI
- Doyle, 2007, Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system, PNAS, 104, 11138, 10.1073/pnas.0703980104 · DOI
- Doyle, 2007, Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity, Nature Structural & Molecular Biology, 14, 114, 10.1038/nsmb1198 · DOI
- Duran, 2017, Comparative analysis of the structure and function of AAA+ motors ClpA, ClpB, and Hsp104: Common threads and disparate functions, Frontiers in Molecular Biosciences, 4, 10.3389/fmolb.2017.00054 · DOI
- Duran, 2018, ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide, Biophysical Chemistry, 242, 6, 10.1016/j.bpc.2018.08.005 · DOI
- Durie, 2018, Escherichia coli Dnak allosterically modulates clpb between high- and low-peptide affinity states, Biochemistry, 57, 3665, 10.1021/acs.biochem.8b00045 · DOI
- Durie, 2019, Hsp104 and potentiated variants can operate as distinct nonprocessive translocases, Biophysical Journal, 116, 1856, 10.1016/j.bpj.2019.03.035 · DOI
- Erzberger, 2006, Evolutionary relationships and structural mechanisms of AAA+ proteins, Annual Review of Biophysics and Biomolecular Structure, 35, 93, 10.1146/annurev.biophys.35.040405.101933 · DOI
- Fischer, 2004, ATP-dependent translocation of proteins along single-stranded DNA: models and methods of analysis of pre-steady state kinetics, Journal of Molecular Biology, 344, 1265, 10.1016/j.jmb.2004.10.004 · DOI
- Fischer, 2004, Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA, Journal of Molecular Biology, 344, 1287, 10.1016/j.jmb.2004.10.005 · DOI
- Gates, 2020, Stairway to translocation: AAA+ motor structures reveal the mechanisms of ATP-dependent substrate translocation, Protein Science, 29, 407, 10.1002/pro.3743 · DOI
- Glover, 1998, Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins, Cell, 94, 73, 10.1016/s0092-8674(00)81223-4 · DOI
- Glynn, 2020, AAA+ proteins: converging mechanisms, diverging functions, Nature Structural & Molecular Biology, 27, 515, 10.1038/s41594-020-0444-2 · DOI
- Goloubinoff, 1999, Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network, PNAS, 96, 13732, 10.1073/pnas.96.24.13732 · DOI
- Hoskins, 2000, Substrate recognition by the ClpA chaperone component of ClpAP protease, The Journal of Biological Chemistry, 275, 35361, 10.1074/jbc.M006288200 · DOI
- Hwang, 2011, Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity, PNAS, 108, 7414, 10.1073/pnas.1017672108 · DOI
- Hwang, 2014, Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions, Chemical Society Reviews, 43, 1221, 10.1039/c3cs60201j · DOI
- Ingram, 2021, Multi-start Evolutionary Nonlinear OpTimizeR (MENOTR): A hybrid parameter optimization toolbox, Biophysical Chemistry, 279, 10.1016/j.bpc.2021.106682 · DOI
- Kenniston, 2003, Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine, Cell, 114, 511, 10.1016/s0092-8674(03)00612-3 · DOI
- Kim, 2002, Interaction of the DnaK and DnaJ chaperone system with a native substrate, P1 RepA, The Journal of Biological Chemistry, 277, 44778, 10.1074/jbc.M206176200 · DOI
- Li, 2015, Examination of polypeptide substrate specificity for Escherichia coli ClpB, Proteins, 83, 117, 10.1002/prot.24710 · DOI
- Li, 2015, Escherichia coli ClpB is a non-processive polypeptide translocase, The Biochemical Journal, 470, 39, 10.1042/BJ20141457 · DOI
- Lin, 2015, Examination of the dynamic assembly equilibrium for E. coli ClpB, Proteins, 83, 2008, 10.1002/prot.24914 · DOI
- Lin, 2016, Examination of clpb quaternary structure and linkage to nucleotide binding, Biochemistry, 55, 1758, 10.1021/acs.biochem.6b00122 · DOI
- Lin, 2022, AAA+ proteins: one motor, multiple ways to work, Biochemical Society Transactions, 50, 895, 10.1042/BST20200350 · DOI
- Lucius, 2003, General methods for analysis of sequential “n-step” kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding, Biophysical Journal, 85, 2224, 10.1016/S0006-3495(03)74648-7 · DOI
- Mazal, 2019, Tunable microsecond dynamics of an allosteric switch regulate the activity of a AAA+ disaggregation machine, Nature Communications, 10, 10.1038/s41467-019-09474-6 · DOI
- Mazal, 2021, Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation, Science Advances, 7, 10.1126/sciadv.abg4674 · DOI
- Miller, 2013, E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP, Journal of Molecular Biology, 425, 2795, 10.1016/j.jmb.2013.04.019 · DOI
- Motohashi, 1999, Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones, PNAS, 96, 7184, 10.1073/pnas.96.13.7184 · DOI
- Nagy, 2004, Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study, Thermochimica Acta, 410, 161, 10.1016/S0040-6031(03)00397-6 · DOI
- Neuwald, 1999, AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes, Genome Research, 9, 27, 10.1101/gr.9.1.27 · DOI
- Oberhauser, 2001, Stepwise unfolding of titin under force-clamp atomic force microscopy, PNAS, 98, 468, 10.1073/pnas.98.2.468 · DOI
- Oguchi, 2012, A tightly regulated molecular toggle controls AAA+ disaggregase, Nature Structural & Molecular Biology, 19, 1338, 10.1038/nsmb.2441 · DOI
- Ogura, 2001, AAA+ superfamily ATPases: common structure--diverse function, Genes to Cells, 6, 575, 10.1046/j.1365-2443.2001.00447.x · DOI
- Olivares, 2014, Mechanochemical basis of protein degradation by a double-ring AAA+ machine, Nature Structural & Molecular Biology, 21, 871, 10.1038/nsmb.2885 · DOI
- Olivares, 2017, Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines, PNAS, 114, E6306, 10.1073/pnas.1707794114 · DOI
- Olivares, 2018, Mechanical protein unfolding and degradation, Annual Review of Physiology, 80, 413, 10.1146/annurev-physiol-021317-121303 · DOI
- Rajendar, 2010, Molecular mechanism of polypeptide translocation catalyzed by the Escherichia coli ClpA protein translocase, Journal of Molecular Biology, 399, 665, 10.1016/j.jmb.2010.03.061 · DOI
- Rashid, 2019, Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation, Nature Communications, 10, 10.1038/s41467-019-10137-9 · DOI
- Reid, 2001, ClpA mediates directional translocation of substrate proteins into the ClpP protease, PNAS, 98, 3768, 10.1073/pnas.071043698 · DOI
- Rizo, 2019, Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase, Nature Communications, 10, 10.1038/s41467-019-10150-y · DOI
- Schrodinger LLC. 2015a. The axpymol molecular graphics plugin for microsoft powerpoint. AxPyMOL. Version 2.5.
- Schrodinger LLC. 2015b. The pymol molecular graphics system. PyMOL. Version 2.5.
- Seidman, 2001, Introduction of plasmid DNA into cells, Current Protocols in Molecular Biology, Chapter 1, 10.1002/0471142727.mb0108s37 · DOI
- Seraphim, 2020, AAA+ proteins, Current Biology, 30, R251, 10.1016/j.cub.2020.01.044 · DOI
- Sharan, 2009, Recombineering: a homologous recombination-based method of genetic engineering, Nature Protocols, 4, 206, 10.1038/nprot.2008.227 · DOI
- Sharma, 2004, Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators, The Journal of Biological Chemistry, 279, 6027, 10.1074/jbc.M310917200 · DOI
- Shorter, 2008, Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions, The EMBO Journal, 27, 2712, 10.1038/emboj.2008.194 · DOI
- Shorter, 2018, Editorial: The role of AAA+ proteins in protein repair and degradation, Frontiers in Molecular Biosciences, 5, 10.3389/fmolb.2018.00085 · DOI
- Snider, 2008, The AAA+ superfamily of functionally diverse proteins, Genome Biology, 9, 10.1186/gb-2008-9-4-216 · DOI
- Weaver, 2017, Avidity for polypeptide binding by nucleotide-bound Hsp104 Structures, Biochemistry, 56, 2071, 10.1021/acs.biochem.7b00225 · DOI
- Weber-Ban, 1999, Global unfolding of a substrate protein by the Hsp100 chaperone ClpA, Nature, 401, 90, 10.1038/43481 · DOI
- Weibezahn, 2004, Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB, Cell, 119, 653, 10.1016/j.cell.2004.11.027 · DOI
- Yagawa, 2010, Structural basis for unfolding pathway-dependent stability of proteins: vectorial unfolding versus global unfolding, Protein Science, 19, 693, 10.1002/pro.346 · DOI
- Zolkiewski, 1999, ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli, The Journal of Biological Chemistry, 274, 28083, 10.1074/jbc.274.40.28083 · DOI
Metadata
- Authors
- Banwait, J. K., Islam, L., & Lucius, A. L.
- Affiliations
- University of Alabama at Birmingham (US)
- Corresponding author
- Lucius, Aaron L
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2024-10-07
- Volume
- 13
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC11458177
- OpenAlex
- W4400416170
- Cited by
- 6
- References
- 1
- Citations by year
- 2026: 3 · 2025: 3
- Type
- posted content
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- National Science Foundation (MCB-1412624, OAC-1541310)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Keywords
- ClpB, Hsp104, protein unfolding, single-turnover kinetics, stopped-flow fluorescence, protein aggregation, ATP hydrolysis, translocation mechanism, Escherichia coli, Saccharomyces cerevisiae
- MeSH terms
- Escherichia coli, Heat-Shock Proteins, Kinetics, Saccharomyces cerevisiae Proteins, Escherichia coli Proteins, Endopeptidase Clp, Protein Unfolding
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