scholarly journals Theory of Allosteric Regulation in Hsp70 Molecular Chaperones

QRB Discovery ◽  
2020 ◽  
Vol 1 ◽  
Author(s):  
Wayne A. Hendrickson
Keyword(s):  
Molecular Chaperones ◽  
Atp Hydrolysis ◽  
Binding Domain ◽  
Substrate Affinity ◽  
Peptide Substrates ◽  
Cellular Processes ◽  
Slow Kinetics ◽  
Shock Proteins ◽  
Kinetics Of ◽  
Substrate Binding Domain

AbstractHeat-shock proteins of 70 kDa (Hsp70s) are ubiquitous molecular chaperones that function in protein folding as well as other vital cellular processes. They bind and hydrolyze ATP in a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides in a substrate-binding domain (SBD). However, the molecular mechanism for this allosteric action has remained unclear. Here, we develop and experimentally quantify a theoretical model for Hsp70 allostery based on equilibria among Hsp70 conformational states. We postulate that, when bound to ATP, Hsp70 is in equilibrium between a restraining state (R) that restricts ATP hydrolysis and binds peptides poorly, if at all, and a stimulating state (S) that hydrolyzes ATP relatively rapidly and has high intrinsic substrate affinity but rapid binding kinetics; after the hydrolysis to ADP, NBD and SBD disengage into an uncoupled state (U) that binds peptide substrates tightly, but now with slow kinetics of exchange.

scholarly journals The Link That Binds: The Linker of Hsp70 as a Helm of the Protein’s Function

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 543 ◽  
Author(s):  
Chakafana ◽  
Zininga ◽  
Shonhai
Keyword(s):  
Protein Aggregation ◽  
Molecular Chaperones ◽  
Atp Hydrolysis ◽  
Sequence Data ◽  
Substrate Binding ◽  
Binding Domain ◽  
Nucleotide Exchange ◽  
Hsp70 Family ◽  
Nucleotide Exchange Factors ◽  
Functional Features

The heat shock 70 (Hsp70) family of molecular chaperones plays a central role in maintaining cellular proteostasis. Structurally, Hsp70s are composed of an N-terminal nucleotide binding domain (NBD) which exhibits ATPase activity, and a C-terminal substrate binding domain (SBD). The binding of ATP at the NBD and its subsequent hydrolysis influences the substrate binding affinity of the SBD through allostery. Similarly, peptide binding at the C-terminal SBD stimulates ATP hydrolysis by the N-terminal NBD. Interdomain communication between the NBD and SBD is facilitated by a conserved linker segment. Hsp70s form two main subgroups. Canonical Hsp70 members generally suppress protein aggregation and are also capable of refolding misfolded proteins. Hsp110 members are characterized by an extended lid segment and their function tends to be largely restricted to suppression of protein aggregation. In addition, the latter serve as nucleotide exchange factors (NEFs) of canonical Hsp70s. The linker of the Hsp110 family is less conserved compared to that of the canonical Hsp70 group. In addition, the linker plays a crucial role in defining the functional features of these two groups of Hsp70. Generally, the linker of Hsp70 is quite small and varies in size from seven to thirteen residues. Due to its small size, any sequence variation that Hsp70 exhibits in this motif has a major and unique influence on the function of the protein. Based on sequence data, we observed that canonical Hsp70s possess a linker that is distinct from similar segments present in Hsp110 proteins. In addition, Hsp110 linker motifs from various genera are distinct suggesting that their unique features regulate the flexibility with which the NBD and SBD of these proteins communicate via allostery. The Hsp70 linker modulates various structure-function features of Hsp70 such as its global conformation, affinity for peptide substrate and interaction with co-chaperones. The current review discusses how the unique features of the Hsp70 linker accounts for the functional specialization of this group of molecular chaperones.

scholarly journals Kinetics of the ATP Hydrolysis Cycle of the Nucleotide-binding Domain of Mdl1 Studied by a Novel Site-specific Labeling Technique

2005 ◽  
Vol 281 (9) ◽  
pp. 5694-5701 ◽  
Author(s):  
Chris van der Does ◽  
Chiara Presenti ◽  
Katrin Schulze ◽  
Stephanie Dinkelaker ◽  
Robert Tampé
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Site Specific ◽  
Kinetics Of

scholarly journals Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Paolo De Los Rios ◽  
Alessandro Barducci
Keyword(s):  
Energy Consumption ◽  
Molecular Chaperones ◽  
Atp Hydrolysis ◽  
Fundamental Issue ◽  
Physiological Conditions ◽  
Substrate Complex ◽  
Biochemical Studies ◽  
Shock Proteins ◽  
Non Equilibrium

70-kDa Heat shock proteins are ATP-driven molecular chaperones that perform a myriad of essential cellular tasks. Although structural and biochemical studies have shed some light on their functional mechanism, the fundamental issue of the role of energy consumption, due to ATP-hydrolysis, has remained unaddressed. Here we establish a clear connection between the non-equilibrium nature of Hsp70, due to ATP hydrolysis, and the determining feature of its function, namely its high affinity for its substrates. Energy consumption can indeed decrease the dissociation constant of the chaperone-substrate complex by several orders of magnitude with respect to an equilibrium scenario. We find that the biochemical requirements for observing such ultra-affinity coincide with the physiological conditions in the cell. Our results rationalize several experimental observations and pave the way for further analysis of non-equilibrium effects underlying chaperone functions.

Direct inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free state

2010 ◽  
Vol 66 (3) ◽  
pp. 223-232 ◽  
Author(s):  
Meiri Shida ◽  
Akihiko Arakawa ◽  
Ryohei Ishii ◽  
Seiichiro Kishishita ◽  
Tetsuo Takagi ◽  
...  
Keyword(s):  
Closed Form ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Free State ◽  
Open Form ◽  
Nucleotide Binding Domain ◽  
Cellular Processes ◽  
Direct Inter ◽  
Shock Proteins ◽  
Hydrophobic Helix

The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5′-(β,γ-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the `closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.

scholarly journals Interdomain communication in the molecular chaperone DnaK

2003 ◽  
Vol 369 (3) ◽  
pp. 627-634 ◽  
Author(s):  
Wanjiang HAN ◽  
Philipp CHRISTEN
Keyword(s):  
Substrate Binding ◽  
Peptide Binding ◽  
Binding Domain ◽  
Intrinsic Fluorescence ◽  
Tryptophan Residue ◽  
Atpase Domain ◽  
Chaperone Dnak ◽  
The One ◽  
Kinetics Of ◽  
Substrate Binding Domain

DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP·DnaK and GrpE-liganded ADP·DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP·DnaK and ADP·DnaK·GrpE and rendered their spectra similar to the spectrum of ADP·DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP·DnaK on the one hand, and to ATP·DnaK or ADP·DnaK·GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.

scholarly journals The HSP Immune Network in Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Zarema Albakova ◽  
Yana Mangasarova
Keyword(s):  
Immune Responses ◽  
Molecular Chaperones ◽  
Epithelial Mesenchymal Transition ◽  
Tumor Development ◽  
Mesenchymal Transition ◽  
Cellular Processes ◽  
Unfolded Protein ◽  
Intracellular Activities ◽  
Shock Proteins ◽  
Protein Response

Heat shock proteins are molecular chaperones which support tumor development by regulating various cellular processes including unfolded protein response, mitochondrial bioenergetics, apoptosis, autophagy, necroptosis, lipid metabolism, angiogenesis, cancer cell stemness, epithelial-mesenchymal transition and tumor immunity. Apart from their intracellular activities, HSPs have also distinct extracellular functions. However, the role that HSP chaperones play in the regulation of immune responses inside and outside the cell is not yet clear. Herein, we explore the intracellular and extracellular immunologic functions of HSPs in cancer. A broader understanding of how HSPs modulate immune responses may provide critical insights for the development of effective immunotherapies.

Designing Specific HSP70 Substrate Binding Domain Inhibitor for Perturbing Protein Folding Pathways to Inhibit Cancer Mechanism

2020 ◽  
Vol 20 ◽  
Author(s):  
Kübra A. Coşkun ◽  
İrfan Koca ◽  
Mehmet Gümüş ◽  
Yusuf Tutar
Keyword(s):  
In Silico ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Binding Domain ◽  
Hydrolysis Rate ◽  
Inhibitor Molecule ◽  
In Silico Docking ◽  
Substrate Protein ◽  
Presence And Absence ◽  
Substrate Binding Domain

Background: HSP70 is a survival factor for tumor cells in transformation and in tumor progression as well as in anti-apoptotic response. Objective: Several inhibitors targeting HSP70 ATPase function displayed off-target affect but PES which targets HSP70 substrate binding domain prevents tumor cell survival prominently. However, PES may not bind HSP70 in the absence of nucleotide. This research aimed to design a unique inhibitor molecule that work both in the presence and absence of nucleotides to amplify inhibition. Methods: A set of chimeric coumarine-pyrazole derivatives determined by in silico techniques and synthesized to elucidate their inhibitory effects. Cell viability experiments displayed KBR1307 as the most efficient inhibitor. A set of characterization experiments performed, and results compared to that of PES agent. Binding constant, ATP hydrolysis rate, and percent aggregation determined in the presence and absence of inhibitors. Results: In silico docking experiments showed that only KBR1307 bind HSP70 substrate binding domain and interact with cochaperone interface. Binding experiments indicated that KBR1307 bind HSP70 both in the presence and absence of nucleotides but PES not. Both inhibitors significantly lower HSP70 ATPase activity and substrate protein disaggregation activity. However, KBR1307 display lower IC50 value at MCF-7 cell line compared to PES. Both inhibitors do not alter HSP70 secondary structure composition and overall stability. Conclusion: KBR1307 effectively inhibits HSP70 compared to PES and provides promising template for novel anticancer drug development.

Insights Into the Molecular Mechanisms of Actin Dynamics: A Multiscale Modeling Approach

2011 ◽  
Author(s):  
Tamara C. Bidone ◽  
Marco A. Deriu ◽  
Giacomo Di Benedetto ◽  
Diana Massai ◽  
Umberto Morbiducci
Keyword(s):  
Cell Migration ◽  
Self Assembly ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Multiple Equilibrium ◽  
Actin Dynamics ◽  
Cellular Processes ◽  
Kinetics Of ◽  
Binding Loop ◽  
Conformational Shifts

Actin dynamics, which is at the basis of many fundamental cellular processes as cell migration [1], is governed by the self-assembly and disassembly of actin monomers (G-actin) that, in turn, are determined by the kinetics of ATP hydrolysis and by the local concentrations of Mg2+ and Ca2+ [2]. During cell migration, interactions of the actin filaments (F-actin) with different nucleotide-cation complexes induce local topological rearrangements, because the filament building G-actins undergo conformational shifts between multiple equilibrium states separated by low-energy barriers. For example, the structural rearrangements of the DNase-I binding loop (residues 38–52) in subdomain 2 are driven by ATP hydrolysis and the changes in the conformation of subdomain 4 are induced by the presence of a tightly-bound Mg2+ or Ca2+ ion (Figure 1a). These conformational shifts alter the cross-linking between monomers, varying the contact surfaces among adjacent inter- and intrasubdomains of G-actin, and reflect on the overall properties of F-actin.

scholarly journals Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes

1991 ◽  
Vol 277 (2) ◽  
pp. 521-526 ◽  
Author(s):  
Q L Ying ◽  
A R Rinehart ◽  
S R Simon ◽  
J C Cheronis
Keyword(s):  
Binding Site ◽  
Ursolic Acid ◽  
Substrate Binding ◽  
Binding Domain ◽  
Glycyrrhetic Acid ◽  
Pentacyclic Triterpenoids ◽  
Peptide Substrates ◽  
Leucocyte Elastase ◽  
Hydrolysis Of ◽  
Substrate Binding Domain

Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids.

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