The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

English, Charles A.; Sherman, Woody; Meng, Wenli; Gierasch, Lila M.

doi:10.1074/jbc.m117.789313

Cited by 54 publications

(68 citation statements)

References 54 publications

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“…In our group we have studied allosterism in both directions: first, from the NBD to the SBD, studying BiP mechanical properties at the single molecule level with optical tweezer manipulation, and now in this work from the SBD to the NBD with ensemble measurements with nano‐rheological experimental setup. This study has shown that there exists a communication between both domains, as when peptide is bound in the SBD, the affinity for ADP bound in the NBD is modified (evidenced by the drop in K D value), demonstrating that SBD is allosterically coupled to NBD, as described in previous studies . This was expectable, considering that it was reported also that peptide binding accelerates BiP's ATPase activity .…”

Section: Discussionsupporting

confidence: 80%

“…This study has shown that there exists a communication between both domains, as when peptide is bound in the SBD, the affinity for ADP bound in the NBD is modified (evidenced by the drop in K D value), demonstrating that SBD is allosterically coupled to NBD, as described in previous studies. 30 This was expectable, considering that it was reported also that peptide binding accelerates BiP's ATPase activity. 31 However, it has not been tested before that the mechanical response of BiP upon ligand binding, can modulate the function of both binding domains particularly operating via changes in its internal viscosity.…”

Section: K D 'S Determination For Bip Nucleotides and Allosterism Betmentioning

confidence: 92%

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Mechanical properties of BiP protein determined by nano‐rheology

et al. 2018

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Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

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Section: Discussionsupporting

confidence: 80%

Section: K D 'S Determination For Bip Nucleotides and Allosterism Betmentioning

confidence: 92%

Mechanical properties of BiP protein determined by nano‐rheology

et al. 2018

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“…Though no model for the mechanistic role of the D HPD -R NBD interaction per se has been previously put forth, the functional connection between these residues is well supported by genetic and biochemical data. Alteration of either R NBD or residues interacting with it (D SBD and D Lk ) 10, 11,14,17,34,38 disrupt allosteric communication between domains, resulting in a somewhat increased basal activity and ineffective stimulation by its JDP partner, DnaJ in the case of DnaK 14 and Hsc20 in the case of Ssc1 ( Supplementary Fig. S4).…”

Section: Discussionmentioning

confidence: 99%

“…A critical segment of this interface is formed by interactions at the base of the NBD with the linker and SBDb (called NBD/SBDb,linker throughout) 5,6, 13 . The linker plays an important role, as its interaction with the NBD is key to stimulation of ATPase activity [14][15][16][17] . A transient allosterically active intermediate poised for hydrolysis of ATP, often referred to as the "allosterically active state", has been observed in the presence of a peptide substrate -NBD/SBD contacts are largely absent, but the linker remains bound to the NBD 10, 18 .…”

Section: Introductionmentioning

confidence: 99%

Two-step mechanism of J-domain action in driving Hsp70 function

Tomiczek

Delewski

Nierzwicki

et al. 2020

Preprint

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J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface -thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition -via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage. are able to discriminate amongst their multiple JDP partners, thus enabling evolution of complex JDP/Hsp70 interaction networks. ResultsStructural model of the Hsc20-Ssq1 complex. To obtain a structural model of the JDP-Hsp70 complex formed by Hsc20 and Ssq1, we combined protein-protein docking with all-atom molecular dynamics (MD) simulations ( Supplementary Fig. S1). This approach revealed a dominant bound state that accounted for 56% of the bound population ( Fig. 1, Supplementary Fig. S2a). No other bound state accounted for more than 6% of the ensemble; these minor states rapidly interconverted and had a clear tendency to converge to the most populated bound state ( Supplementary Fig. S2b). The binding mode in this dominant state closely resembles the recently published X-ray structure of DnaJ's J-domain in complex with DnaK (DnaJ JD -DnaK) 22 . Helices II and III are oriented very similarly in the two complexes -with a backbone root mean square deviation (RMSD) of 4.17 Å ( Supplementary Fig. S3). More specifically, the J-domains bind at the NBD/SBDb,linker interface such that helix II predominantly interacts with the b-sheet region of NBD subdomain IIa and helix III with SBDb (Fig. 1). The HPD residues are positioned nearly identically in the two complexes, towards R167/R207 of NBD subdomain Ia in DnaK/Ssq1 (referred to as R NBD throughout), the arginine that previous analyses of DnaK established as important for allosteric transitions 14,20,21 . D HPD interferes in Hsp70 interdomain interactions by perturbing critical R NBD contacts.To address the question of the mechanism of J-domain action, we employed MD simulations that, unlike most other methods, allow studying of transient r...

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“…The SBD and NBD remain attached through a short linker that has been implicated in modulating the domain interactions. In the ATP bound state, the linker has been observed to bind the NBD facilitating the docked conformation (2,3). Disruption of the NBD-linker interaction has been suggested to promote the undocked conformation associated with ATP-hydrolysis (4).…”

Section: Introductionmentioning

confidence: 99%

Structural influence of the conserved Hsp40 HPD tripeptide on Hsp70 chaperone function

Mallapre

Bascos

2020

Preprint

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The control of Hsp70 functions has been related to the modulation of ATP hydrolysis and substrate capture by Hsp40. Structural and biophysical analyses of Hsp40 variants and their interactions with Hsp70 have identified key residues for this functional control mechanism. Conserved residues in both Hsp40 and Hsp70 have revealed conserved interactions that link Hsp40 binding to the catalytic residues within Hsp70. The current work investigates the effect of documented J-domain dysfunctional mutations (i.e. D35N, H33Q) on the described interaction linkage. Molecular dynamics simulations were used to compare the persistence of individual bond types (i.e. H-bonds, salt bridges, hydrophobic interactions) between Hsp70 and the bound forms of functional and dysfunctional Hsp40 variants. The generated data suggests the involvement of both direct and allosteric effects for the tested mutations. The observed changes relate mutations in the conserved HPD tripeptide of Hsp40 to alterations in the interaction network that induces Hsp70 chaperone functions.The significance of the work may be summarized as follows. First, the interaction network for the simulated systems were observed to be different from one previously proposed for a disulfide linked complex (9). This may be attributed to altered residue movement and interactions without the restrictions set by the disulfide link. These results support the use of in silico methods to refine investigations of molecular contacts, particularly for systems, whose in vitro structural elucidation are difficult to achieve without modifications.Second, key interactions for intermolecular and intramolecular contacts were observed within a short simulation time (0.1 ns) matched those from much longer runs (500 ns) (4). This result highlights the possibility of identifying key interactions with relatively low computational cost.

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The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

Cited by 54 publications

References 54 publications

Mechanical properties of BiP protein determined by nano‐rheology

Mechanical properties of BiP protein determined by nano‐rheology

Two-step mechanism of J-domain action in driving Hsp70 function

Structural influence of the conserved Hsp40 HPD tripeptide on Hsp70 chaperone function

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