Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90

AbstractThe heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

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Conformational dynamics of the molecular chaperone Hsp90

Quarterly Reviews of Biophysics ◽

10.1017/s0033583510000314 ◽

2011 ◽

Vol 44 (2) ◽

pp. 229-255 ◽

Cited By ~ 186

Author(s):

Kristin A. Krukenberg ◽

Timothy O. Street ◽

Laura A. Lavery ◽

David A. Agard

Keyword(s):

Molecular Chaperone ◽

Structural Changes ◽

Atp Hydrolysis ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Post Translational Modification ◽

Regulatory Processes ◽

Structural Methods

AbstractThe ubiquitous molecular chaperone Hsp90 makes up 1–2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo–ATP–ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90, as well as the roles that nucleotide, co-chaperones, post-translational modification and substrates play. This view of Hsp90's conformational dynamics was enabled by the use of multiple complementary structural methods including, crystallography, small-angle X-ray scattering (SAXS), electron microscopy, Förster resonance energy transfer (FRET) and NMR. Finally, we discuss the effects of Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.

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Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858)

Journal of Bacteriology ◽

10.1128/jb.00905-15 ◽

2016 ◽

Vol 198 (10) ◽

pp. 1521-1533 ◽

Cited By ~ 4

Author(s):

Mihaela-Carmen Unciuleac ◽

Paul C. Smith ◽

Stewart Shuman

Keyword(s):

Crystal Structure ◽

Atpase Activity ◽

Active Site ◽

Mycobacterium Smegmatis ◽

Tertiary Structure ◽

Biochemical Characterization ◽

Atp Hydrolysis ◽

Content Type ◽

Atpase Reaction ◽

Aaa Protein

ABSTRACTAAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of aMycobacterium smegmatisAAA protein Msm0858, the ortholog ofMycobacterium tuberculosisRv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer.IMPORTANCERecent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

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Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720448115 ◽

2018 ◽

Vol 115 (13) ◽

pp. 3243-3248 ◽

Cited By ~ 35

Author(s):

Haim Yuval Aviram ◽

Menahem Pirchi ◽

Hisham Mazal ◽

Yoav Barak ◽

Inbal Riven ◽

...

Keyword(s):

Single Molecule ◽

Active Site ◽

Adenylate Kinase ◽

Large Scale ◽

Turnover Rate ◽

Conformational Dynamics ◽

Mutual Orientation ◽

Single Molecule Fret ◽

Domain Motions ◽

Active Site Mutants

The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100–200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

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The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action

Annual Review of Microbiology ◽

10.1146/annurev-micro-032421-035644 ◽

2021 ◽

Vol 75 (1) ◽

Author(s):

Sue Wickner ◽

Thu-Lan Lily Nguyen ◽

Olivier Genest

Keyword(s):

Molecular Chaperone ◽

Conformational Dynamics ◽

Heat Shock Protein 90 ◽

Annual Review ◽

Publication Date ◽

Cellular Functions ◽

Hsp90 Chaperone ◽

Client Proteins ◽

Protein Remodeling ◽

Substrate Proteins

Heat shock protein 90 (Hsp90) is a molecular chaperone that folds and remodels proteins, thereby regulating the activity of numerous substrate proteins. Hsp90 is widely conserved across species and is essential in all eukaryotes and in some bacteria under stress conditions. To facilitate protein remodeling, bacterial Hsp90 collaborates with the Hsp70 molecular chaperone and its cochaperones. In contrast, the mechanism of protein remodeling performed by eukaryotic Hsp90 is more complex, involving more than 20 Hsp90 cochaperones in addition to Hsp70 and its cochaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of bacterial Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70. We describe the universally conserved structure and conformational dynamics of these chaperones and their interactions with one another and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide a framework for understanding the more complex eukaryotic Hsp90 system. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657715 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1202-1208 ◽

Cited By ~ 18

Author(s):

Marianne Kjalke ◽

Julie A Oliver ◽

Dougald M Monroe ◽

Maureane Hoffman ◽

Mirella Ezban ◽

...

Keyword(s):

Tissue Factor ◽

Active Site ◽

Large Scale ◽

Thrombin Generation ◽

Factor Viia ◽

Dependent Manner ◽

Antithrombotic Agent ◽

Before And After ◽

Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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Faculty Opinions recommendation of Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1082926.535884 ◽

2007 ◽

Author(s):

Homer Pearce

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Molecular Chaperone ◽

Heat Shock Protein 90

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Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80529-8 ◽

1993 ◽

Vol 268 (32) ◽

pp. 24323-24329

Author(s):

M.C. O'Brien ◽

D.B. McKay

Keyword(s):

Active Site ◽

Atp Hydrolysis ◽

Chaperone Protein

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Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

International Journal of Molecular Sciences ◽

10.3390/ijms22094769 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4769

Author(s):

Pablo Maturana ◽

María S. Orellana ◽

Sixto M. Herrera ◽

Ignacio Martínez ◽

Maximiliano Figueroa ◽

...

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Catalytic Mechanism ◽

Conformational Dynamics ◽

High Specificity ◽

Arginine Decarboxylase ◽

Space Groups ◽

X Ray Crystallography ◽

High Dynamics ◽

Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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