scholarly journals Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Simon Jenni ◽  
Benjamin M Stinson ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

scholarly journals Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

2019 ◽  
Author(s):  
Xue Fei ◽  
Tristan A. Bell ◽  
Simon Jenni ◽  
Benjamin M. Stinson ◽  
Tania A. Baker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Functional Results ◽  
Structural Features ◽  
Specific Protein ◽  
Power Stroke ◽  
E Coli ◽  
Protein Substrates ◽  
Biophysical Studies ◽  
Sequential Models

SUMMARYClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

scholarly journals Multistep substrate binding and engagement by the AAA+ ClpXP protease

2020 ◽  
Author(s):  
Reuben A. Saunders ◽  
Benjamin M. Stinson ◽  
Tania A. Baker ◽  
Robert T. Sauer
Keyword(s):  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Specific Protein ◽  
Dissociation Kinetics ◽  
E Coli ◽  
Protein Substrates ◽  
Channel Opening ◽  
Initial Substrate ◽  
Domains Of Life ◽  
Initial Binding

AbstractE. coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.SignificanceAAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show that E. coli ClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals The Physics of Entropic Pulling: A Novel Model for the Hsp70 Motor Mechanism

2019 ◽  
Vol 20 (9) ◽  
pp. 2334 ◽  
Author(s):  
Rui Sousa ◽  
Eileen M. Lafer
Keyword(s):  
Experimental Evidence ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Power Stroke ◽  
Brownian Ratchet ◽  
Protein Substrates ◽  
Basic Physics ◽  
Physical Changes ◽  
Novel Model

Hsp70s use ATP to generate forces that disassemble protein complexes and aggregates, and that translocate proteins into organelles. Entropic pulling has been proposed as a novel mechanism, distinct from the more familiar power-stroke and Brownian ratchet models, for how Hsp70s generate these forces. Experimental evidence supports entropic pulling, but this model may not be well understood among scientists studying these systems. In this review we address persistent misconceptions regarding the dynamics of proteins in solution that contribute to this lack of understanding, and we clarify the basic physics of entropic pulling with some simple analogies. We hope that increased understanding of the entropic pulling mechanism will inform future efforts to characterize how Hsp70s function as motors, and how they coordinate with their regulatory cochaperones in mechanochemical cycles that transduce the energy of ATP hydrolysis into physical changes in their protein substrates.

scholarly journals Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation

2018 ◽  
Vol 19 (11) ◽  
pp. 3651 ◽  
Author(s):  
Justin Marsee ◽  
Amy Ridings ◽  
Tao Yu ◽  
Justin Miller
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Unfolding ◽  
Atp Hydrolysis ◽  
Adaptor Protein ◽  
Specific Protein ◽  
Protein Substrates ◽  
Terminal Domain ◽  
Dependent Inhibition ◽  
Protease Substrate

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

scholarly journals Functional interactions between gyrase subunits are optimized in a species-specific manner

2020 ◽  
Vol 295 (8) ◽  
pp. 2299-2312
Author(s):  
Daniela Weidlich ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Dna Supercoiling ◽  
Structural Features ◽  
Subunit Interaction ◽  
Dna Topoisomerase ◽  
Bacterial Dna ◽  
E Coli ◽  
Functional Interactions ◽  
Species Specific ◽  
Gyrase Inhibitors

DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.

scholarly journals Multistep substrate binding and engagement by the AAA+ ClpXP protease

2020 ◽  
Vol 117 (45) ◽  
pp. 28005-28013 ◽  
Author(s):  
Reuben A. Saunders ◽  
Benjamin M. Stinson ◽  
Tania A. Baker ◽  
Robert T. Sauer
Keyword(s):  
Kinetic Data ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Specific Protein ◽  
Stopped Flow ◽  
Dissociation Kinetics ◽  
Protein Substrates ◽  
Channel Opening ◽  
Initial Substrate ◽  
Quenching Assay

Escherichia coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals Unraveling a Force-Generating Allosteric Pathway of Actomyosin Communication Associated with ADP and Pi Release

2020 ◽  
Vol 22 (1) ◽  
pp. 104
Author(s):  
Peter Franz ◽  
Wiebke Ewert ◽  
Matthias Preller ◽  
Georgios Tsiavaliaris
Keyword(s):  
Structural Changes ◽  
Atp Hydrolysis ◽  
Power Stroke ◽  
Duty Ratio ◽  
Prolonged Duration ◽  
Adp Release ◽  
Strong Transition ◽  
Cleft Closure ◽  
Kinetic Investigations ◽  
Allosteric Pathway

The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.

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