Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

Recent experiments, drawing upon single-molecule, solution kinetic and structural techniques, have clarified our mechanistic understanding of class V myosins. The findings of the past two years can be summarized as follows: (1) Myosin V is a highly efficient processive motor, surpassing even conventional kinesin in the distance that individual molecules can traverse. (2) The kinetic scheme underlying ATP turnover resembles those of myosins I and II but with rate constants tuned to favor strong binding to actin. ADP release precedes dissociation from actin and is rate-limiting in the cycle. (3) Myosin V walks in strides averaging ∼36 nm, the long pitch pseudo-repeat of the actin helix, each step coupled to a single ATP hydrolysis. Such a unitary displacement, the largest molecular step size measured to date, is required for a processive myosin motor to follow a linear trajectory along a helical actin track.

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AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation

eLife ◽

10.7554/elife.29428 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Steffen Preissler ◽

Lukas Rohland ◽

Yahui Yan ◽

Ruming Chen ◽

Randy J Read ◽

...

Keyword(s):

Atp Hydrolysis ◽

Inverse Correlation ◽

Rate Limiting Step ◽

Hsp70 Chaperone ◽

Tightly Coupled ◽

Client Proteins ◽

Functional Inactivation ◽

Atp Binding And Hydrolysis ◽

Rate Limiting ◽

J Protein

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP’s activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

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Oxidative phosphorylation. Halide-dependent and halide-independent effects of triorganotin and trioganolead compounds on mitochondrial functions

Biochemical Journal ◽

10.1042/bj1680353 ◽

1977 ◽

Vol 168 (3) ◽

pp. 353-364 ◽

Cited By ~ 96

Author(s):

W N Aldridge ◽

B W Street ◽

D N Skilleter

Keyword(s):

Maximum Rate ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

O2 Uptake ◽

Structure Activity ◽

Triethyl Lead ◽

Mitochondrial Functions ◽

Independent Effects ◽

Beta Hydroxybutyrate ◽

Rate Limiting

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.

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Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

10.1101/2021.12.07.471682 ◽

2021 ◽

Author(s):

Mrinal Shekhar ◽

Chitrak Gupta ◽

Kano Suzuki ◽

Abhishek Singharoy ◽

Takeshi Murata

Keyword(s):

Molecular Motors ◽

Atp Hydrolysis ◽

Structural Information ◽

Hydrolysis Product ◽

X Ray ◽

X Ray Crystallography ◽

Rate Limiting Step ◽

Limiting Step ◽

Dynamics Simulations ◽

Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

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The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity

10.1101/2020.05.03.075523 ◽

2020 ◽

Author(s):

Elaine Kirschke ◽

Zygy Roe-Zurz ◽

Chari Noddings ◽

David Agard

Keyword(s):

Glucocorticoid Receptor ◽

Atp Hydrolysis ◽

Signaling Proteins ◽

Toggle Switch ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Complex Process ◽

The Impact ◽

Rate Limiting

AbstractThe glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This can be accomplished by high non-physiological Bag-1 concentrations, which also inhibit Hsp70:ATP binding. In contrast, in an ATP-hydrolysis dependent process, Hsp90 plays a unique role by kinetically partitioning GR into a state that can bind ligand, but is protected from Hsp70 inactivation, thus allowing GR to be activated by its ligand but still able to re-enter the chaperone cycle. At physiologic concentrations, Bag-1 works synergistically with Hsp90 to accelerate the first rate-limiting step in GR reactivation. The net effect is that the chaperone machinery cyclically dictates the on and off rates for GR ligand, providing a timer controlling the persistence of activated GR.Significance StatementThe glucocorticoid receptor (GR) is an essential transcription regulatory factor. Like many signaling proteins, GR activity is regulated by two essential molecular chaperones: Hsp90 and Hsp70. Functioning like a toggle switch, Hsp70 first inactivates GR, and then Hsp90 reactivates GR in an Hsp90 ATP hydrolysis dependent manner. Here, an intricate set of biochemistry experiments uncover fundamental principles governing how these chaperone systems collaboratively regulate GR activity. While Hsp90 promotes GR release from Hsp70 by modulating Hsp70’s nucleotide state, this occurs independently of Hsp90 ATP hydrolysis. Instead, ATP hydrolysis on Hsp90 facilitates a second essential reactivation step resulting in an Hsp90-bound GR state that protects GR from Hsp70 re-inactivation. A kinetic partitioning model best describes chaperone modulation of GR’s activity.

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A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity

eLife ◽

10.7554/elife.03487 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 28

Author(s):

James R Partridge ◽

Laura A Lavery ◽

Daniel Elnatan ◽

Nariman Naber ◽

Roger Cooke ◽

...

Keyword(s):

Crystal Structure ◽

Temperature Dependence ◽

Atp Hydrolysis ◽

Binding Pocket ◽

Chaperone Activity ◽

Regulatory Function ◽

Protein Homeostasis ◽

Closed State ◽

Higher Eukaryotes ◽

Rate Limiting

Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or ‘strap’ and demonstrate its responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally sensitive kinetic barrier between the apo ‘open’ and ATP-bound ‘closed’ conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments.

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Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

10.1101/2020.05.08.084160 ◽

2020 ◽

Author(s):

William J. Allen ◽

Daniel W. Watkins ◽

Mark S. Dillingham ◽

Ian Collinson

Keyword(s):

Protein Transport ◽

Atp Hydrolysis ◽

Kinetic Modelling ◽

Membrane Transporters ◽

Transport Activity ◽

Natural Tags ◽

Indirect Coupling ◽

Sec System ◽

Rate Limiting ◽

Best Fit

AbstractThe universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity – a prerequisite for understanding how biological systems works – has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, ∼30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient – in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

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Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

10.1101/2020.06.30.180695 ◽

2020 ◽

Cited By ~ 2

Author(s):

Yanxin Liu ◽

Ming Sun ◽

Alexander G. Myasnikov ◽

Daniel Elnatan ◽

Nicolas Delaeter ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Atp Hydrolysis ◽

Activation Mechanism ◽

Client Protein ◽

Protein Maturation ◽

Structural Framework ◽

Cryo Electron Microscopy ◽

Client Proteins ◽

Rate Limiting

AbstractHsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.

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Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010906117 ◽

2020 ◽

Vol 117 (50) ◽

pp. 31808-31816

Author(s):

William J. Allen ◽

Daniel W. Watkins ◽

Mark S. Dillingham ◽

Ian Collinson

Keyword(s):

Amino Acids ◽

Kinetic Modeling ◽

Time Resolution ◽

Protein Transport ◽

Atp Hydrolysis ◽

Transport Proteins ◽

Primary Method ◽

Transport Activity ◽

Sec System ◽

Rate Limiting

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

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