Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation
Abstract
The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Furthermore, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.
- Authors:
-
- Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
- Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Takeda Pharmaceuticals, Cambridge, MA (United States)
- Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Stony Brook Univ., NY (United States)
- Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Stanford Univ., CA (United States)
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- National Inst. of Health
- OSTI Identifier:
- 1430301
- Grant/Contract Number:
- AI-16892
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Volume: 292; Journal Issue: 14; Journal ID: ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular Biology
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; ATP-dependent protease; ATPases associated with diverse cellular activities (AAA); crystal structure; protein engineering; protein turnover; AAA+ protease; mixed hexameric rings; protein unfolding; protein degradation; HslUV
Citation Formats
Baytshtok, Vladimir, Chen, Jiejin, Glynn, Steven E., Nager, Andrew R., Grant, Robert A., Baker, Tania A., and Sauer, Robert T. Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation. United States: N. p., 2017.
Web. doi:10.1074/jbc.M116.768978.
Baytshtok, Vladimir, Chen, Jiejin, Glynn, Steven E., Nager, Andrew R., Grant, Robert A., Baker, Tania A., & Sauer, Robert T. Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation. United States. https://doi.org/10.1074/jbc.M116.768978
Baytshtok, Vladimir, Chen, Jiejin, Glynn, Steven E., Nager, Andrew R., Grant, Robert A., Baker, Tania A., and Sauer, Robert T. Tue .
"Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation". United States. https://doi.org/10.1074/jbc.M116.768978. https://www.osti.gov/servlets/purl/1430301.
@article{osti_1430301,
title = {Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation},
author = {Baytshtok, Vladimir and Chen, Jiejin and Glynn, Steven E. and Nager, Andrew R. and Grant, Robert A. and Baker, Tania A. and Sauer, Robert T.},
abstractNote = {The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Furthermore, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.},
doi = {10.1074/jbc.M116.768978},
journal = {Journal of Biological Chemistry},
number = 14,
volume = 292,
place = {United States},
year = {Tue Feb 21 00:00:00 EST 2017},
month = {Tue Feb 21 00:00:00 EST 2017}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 9 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
AAA+ superfamily ATPases: common structure-diverse function
journal, July 2001
- Ogura, Teru; Wilkinson, Anthony J.
- Genes to Cells, Vol. 6, Issue 7
The AAA+ superfamily of functionally diverse proteins
journal, January 2008
- Snider, Jamie; Thibault, Guillaume; Houry, Walid A.
- Genome Biology, Vol. 9, Issue 4
ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli
journal, July 1999
- Seong, Ihn Sik; Oh, Ji Yeon; Yoo, Soon Ji
- FEBS Letters, Vol. 456, Issue 1
Analysis of the Cooperative ATPase Cycle of the AAA+ Chaperone ClpB from Thermus thermophilus by Using Ordered Heterohexamers with an Alternating Subunit Arrangement
journal, February 2015
- Yamasaki, Takashi; Oohata, Yukiko; Nakamura, Toshiki
- Journal of Biological Chemistry, Vol. 290, Issue 15
An Intersubunit Signaling Network Coordinates ATP Hydrolysis by m-AAA Proteases
journal, September 2009
- Augustin, Steffen; Gerdes, Florian; Lee, Sukyeong
- Molecular Cell, Vol. 35, Issue 5
Mutational studies on HslU and its docking mode with HslV
journal, December 2000
- Song, H. K.; Hartmann, C.; Ramachandran, R.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 26
Crystal and Solution Structures of an HslUV Protease–Chaperone Complex
journal, November 2000
- Sousa, Marcelo C.; Trame, Christine B.; Tsuruta, Hiro
- Cell, Vol. 103, Issue 4
Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine
journal, April 2013
- Stinson, Benjamin M.; Nager, Andrew R.; Glynn, Steven E.
- Cell, Vol. 153, Issue 3
Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX
journal, April 2015
- Stinson, Benjamin M.; Baytshtok, Vladimir; Schmitz, Karl R.
- Nature Structural & Molecular Biology, Vol. 22, Issue 5
Asymmetric Nucleotide Transactions of the HslUV Protease
journal, July 2008
- Yakamavich, Joseph A.; Baker, Tania A.; Sauer, Robert T.
- Journal of Molecular Biology, Vol. 380, Issue 5
Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine
journal, May 2012
- Glynn, Steven E.; Nager, Andrew R.; Baker, Tania A.
- Nature Structural & Molecular Biology, Vol. 19, Issue 6
MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009
- Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm
journal, May 2012
- Lobstein, Julie; Emrich, Charlie A.; Jeans, Chris
- Microbial Cell Factories, Vol. 11, Issue 1
Stochastic but Highly Coordinated Protein Unfolding and Translocation by the ClpXP Proteolytic Machine
journal, July 2014
- Cordova, Juan Carlos; Olivares, Adrian O.; Shin, Yongdae
- Cell, Vol. 158, Issue 3
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
Nucleotide-Dependent Conformational Changes in a Protease-Associated ATPase HslU
journal, November 2001
- Wang, J.; Song, J. J.; Seong, I. S.
- Structure, Vol. 9, Issue 11
ClpXP, an ATP-powered unfolding and protein-degradation machine
journal, January 2012
- Baker, Tania A.; Sauer, Robert T.
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1823, Issue 1
The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation: The I Domain Coordinates Substrate Binding and ATP Hydrolysis
journal, January 2012
- Sundar, Shankar; Baker, Tania A.; Sauer, Robert T.
- Protein Science, Vol. 21, Issue 2
Nucleotide-dependent substrate recognition by the AAA+ HslUV protease
journal, February 2005
- Burton, Randall E.; Baker, Tania A.; Sauer, Robert T.
- Nature Structural & Molecular Biology, Vol. 12, Issue 3
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
Asymmetric Interactions of ATP with the AAA+ ClpX6 Unfoldase: Allosteric Control of a Protein Machine
journal, July 2005
- Hersch, Greg L.; Burton, Randall E.; Bolon, Daniel N.
- Cell, Vol. 121, Issue 7
Stepwise Unfolding of a β Barrel Protein by the AAA+ ClpXP Protease
journal, October 2011
- Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.
- Journal of Molecular Biology, Vol. 413, Issue 1
The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears
journal, October 2013
- Sen, Maya; Maillard, Rodrigo A.; Nyquist, Kristofor
- Cell, Vol. 155, Issue 3
Crystal Structures of the HslVU Peptidase–ATPase Complex Reveal an ATP-Dependent Proteolysis Mechanism
journal, February 2001
- Wang, J.; Song, J. J.; Franklin, M. C.
- Structure, Vol. 9, Issue 2
Crystal Structure of HslUV Complexed with a Vinyl Sulfone Inhibitor: Corroboration of a Proposed Mechanism of Allosteric Activation of HslV by HslU
journal, May 2002
- Sousa, Marcelo C.; Kessler, Benedikt M.; Overkleeft, Herman S.
- Journal of Molecular Biology, Vol. 318, Issue 3
Mechanism of DNA translocation in a replicative hexameric helicase
journal, July 2006
- Enemark, Eric J.; Joshua-Tor, Leemor
- Nature, Vol. 442, Issue 7100
Disulfide by DesignTM: a computational method for the rational design of disulfide bonds in proteins
journal, September 2003
- Dombkowski, A. A.
- Bioinformatics, Vol. 19, Issue 14
A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis
journal, October 2016
- Baytshtok, Vladimir; Fei, Xue; Grant, Robert A.
- Structure, Vol. 24, Issue 10
Effects of the Cys Mutations on Structure and Function of the ATP-dependent HslVU Protease in Escherichia coli : THE CYS
journal, September 1998
- Yoo, Soon Ji; Kim, Hyun Hee; Shin, Dong Hun
- Journal of Biological Chemistry, Vol. 273, Issue 36
Structure of Haemophilus influenzae HslU protein in crystals with one-dimensional disorder twinning
journal, July 2001
- Trame, Christine B.; McKay, David B.
- Acta Crystallographica Section D Biological Crystallography, Vol. 57, Issue 8
Enzymatic and Structural Similarities between the Escherichia coli ATP-dependent Proteases, ClpXP and ClpAP
journal, May 1998
- Grimaud, Regis; Kessel, Martin; Beuron, Fabienne
- Journal of Biological Chemistry, Vol. 273, Issue 20
Structure and Reactivity of an Asymmetric Complex between HslV and I-domain Deleted HslU, a Prokaryotic Homolog of the Eukaryotic Proteasome
journal, July 2003
- Kwon, Ae-Ran; Kessler, Benedikt M.; Overkleeft, Herman S.
- Journal of Molecular Biology, Vol. 330, Issue 2
p22 Arc Repressor: Folding Kinetics of a Single-Domain, Dimeric Protein
journal, February 1994
- Milla, Marcos E.; Sauer, Robert T.
- Biochemistry, Vol. 33, Issue 5
Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase
journal, September 2013
- Beckwith, Robyn; Estrin, Eric; Worden, Evan J.
- Nature Structural & Molecular Biology, Vol. 20, Issue 10
The structures of HslU and the ATP-dependent protease HslU–HslV
journal, February 2000
- Bochtler, Matthias; Hartmann, Claudia; Song, Hyun Kyu
- Nature, Vol. 403, Issue 6771
Mechanisms of Conformational Change for a Replicative Hexameric Helicase of SV40 Large Tumor Antigen
journal, October 2004
- Gai, Dahai; Zhao, Rui; Li, Dawei
- Cell, Vol. 119, Issue 1
The C-terminal Tails of HslU ATPase Act as a Molecular Switch for Activation of HslV Peptidase
journal, May 2002
- Seong, Ihn Sik; Kang, Min Suk; Choi, Min Kyung
- Journal of Biological Chemistry, Vol. 277, Issue 29
Structural basis for intersubunit signaling in a protein disaggregating machine
journal, July 2012
- Biter, A. B.; Lee, S.; Sung, N.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 31
Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine
journal, November 2009
- Glynn, Steven E.; Martin, Andreas; Nager, Andrew R.
- Cell, Vol. 139, Issue 4
Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines
journal, October 2005
- Martin, Andreas; Baker, Tania A.; Sauer, Robert T.
- Nature, Vol. 437, Issue 7062
Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY)
journal, May 2002
- Ramachandran, R.; Hartmann, C.; Song, H. K.
- Proceedings of the National Academy of Sciences, Vol. 99, Issue 11
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
Multiple Sequence Signals Direct Recognition and Degradation of Protein Substrates by the AAA+ Protease HslUV
journal, October 2010
- Sundar, Shankar; McGinness, Kathleen E.; Baker, Tania A.
- Journal of Molecular Biology, Vol. 403, Issue 3
Works referencing / citing this record:
Proteolysis mediated by the membrane‐integrated ATP‐dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates
journal, May 2019
- Yang, Yiqing; Gunasekara, Mihiravi; Muhammednazaar, Shaima
- Protein Science
Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex
journal, January 2020
- Jessop, Matthew; Arragain, Benoit; Miras, Roger
- Communications Biology, Vol. 3, Issue 1
New tools for automated high-resolution cryo-EM structure determination in RELION-3
journal, November 2018
- Zivanov, Jasenko; Nakane, Takanori; Forsberg, Björn O.
- eLife, Vol. 7