scholarly journals Single-molecule spectroscopy reveals dynamic allostery mediated by the substrate-binding domain of a AAA+ machine

2020 ◽  
Author(s):  
Marija Iljina ◽  
Hisham Mazal ◽  
Pierre Goloubinoff ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Regulatory Pathways ◽  
Time Dynamics ◽  
Binding Domains ◽  
Substrate Binding Domain

AbstractClpB is an ATP-dependent protein disaggregation machine that is activated on demand by co-chaperones and by aggregates caused by heat shock or mutations. The regulation of ClpB’s function is critical, since its persistent activation is toxic in vivo. Each ClpB molecule is composed of an auxiliary N-terminal domain (NTD), an essential regulatory middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains that are responsible for ATP-fuelled substrate threading. The NTD is generally thought to serve as a substrate-binding domain, which is commonly considered to be dispensable for ClpB’s activity, and is not well-characterized structurally due to its high mobility. Here we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB’s NTD and reveal its involvement in novel allosteric interactions. We find that the NTD fluctuates on a microsecond timescale and, unexpectedly, shows little change in conformational dynamics upon binding of a substrate protein. During its fast motion, the NTD makes crucial contacts with the regulatory MD, directly affecting its conformational state and thereby influencing the overall ATPase and unfolding activity of this machine. Moreover, we also show that the NTD mediates signal transduction to the nucleotide-binding domains through conserved residues. The two regulatory pathways revealed here enable the NTD to suppress the MD in the absence of protein substrate, and to limit ATPase and disaggregation activities of ClpB. The use of multiple parallel allosteric pathways involving ultrafast domain motions might be common to AAA+ molecular machines to ensure their fast and reversible activation.

scholarly journals Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

2017 ◽  
Vol 114 (23) ◽  
pp. 6040-6045 ◽  
Author(s):  
Soumit Sankar Mandal ◽  
Dale R. Merz ◽  
Maximilian Buchsteiner ◽  
Ruxandra I. Dima ◽  
Matthias Rief ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Structural Changes ◽  
Substrate Binding ◽  
Protein Structures ◽  
Binding Domain ◽  
Flexible Region ◽  
Structural Parts ◽  
The Individual ◽  
Substrate Binding Domain

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

scholarly journals Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation

2021 ◽  
Author(s):  
Wei Chen ◽  
Wei Lu ◽  
Peter G Wolynes ◽  
Elizabeth A Komives
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Single Molecule ◽  
Bound States ◽  
Conformational Dynamics ◽  
Conformational Ensemble ◽  
Binding Modes ◽  
Loss Of Function ◽  
Binding Domains

Abstract Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

scholarly journals Substrate-binding domain conformational dynamics mediate Hsp70 allostery

2015 ◽  
Vol 112 (22) ◽  
pp. E2865-E2873 ◽  
Author(s):  
Anastasia Zhuravleva ◽  
Lila M. Gierasch
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Binding Domain ◽  
Substrate Binding Site ◽  
Functional Sites ◽  
Binding Interface ◽  
Wide Range ◽  
Binding Cleft ◽  
Substrate Binding Domain

Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and βSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the βSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the βSBD and force disengagement of SBD strand β8 from strand β7, which leads to repacking of a βSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire βSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the βSBD–the NBD binding interface and the substrate-binding site–confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone–client interactions.

scholarly journals Atypical domain communication and domain functions of a Hsp110 chaperone

2017 ◽  
Author(s):  
Vignesh Kumar ◽  
Joshua Jebakumar Peter ◽  
Amin Sagar ◽  
Arjun Ray ◽  
Ashish ◽  
...  
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Nucleotide Exchange ◽  
Dynamic Simulations ◽  
X Ray ◽  
Single Molecule Fret ◽  
Binding Domains ◽  
Nucleotide Exchange Factors ◽  
X Ray Scattering ◽  
Substrate Binding Domain

AbstractHsp110s are well recognized nucleotide exchange factors (NEFs) of Hsp70s, in addition they are implicated in various aspects of cellular proteostasis as discrete chaperones with yet enigmatic molecular mechanism. Stark similarity in domain organization and structure between Hsp110s and Hsp70s, is easily discernible although the nature of domain communication and domain functions of Hsp110s are still puzzling. Here, we report atypical domain communication of yeast Hsp110, Sse1 using single molecule FRET, small angle X-ray scattering measurements (SAXS) and Molecular Dynamic simulations. Our data show that Sse1 lacks typical domain movements as exhibited by Hsp70s, albeit it undergoes unique structural alteration upon nucleotide and substrate binding. Hsp70-like domain-movements can be artificially salvaged in chimeric constructs of Hsp110-Hsp70 although such salvaging proves detrimental for the NEF activity of the protein. Furthermore, we show that substrate binding domain (SBD) of Hsp110, chaperones self, as well as foreign nucleotide binding domains (NBD). Interestingly, the substrate binding specificity of Hsp110 is largely determined by its NBD rather than SBD, the latter being the foremost substrate binding region for Hsp70s.

scholarly journals S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid

2020 ◽  
Vol 295 (24) ◽  
pp. 8302-8324 ◽  
Author(s):  
Jie Yang ◽  
Hong Zhang ◽  
Weibin Gong ◽  
Zhenyan Liu ◽  
Huiwen Wu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cysteine Residues ◽  
Post Translational Modification ◽  
Hsp70 Family ◽  
And Function

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.

scholarly journals An Essential Role for the Substrate-Binding Region of Hsp40s in Saccharomyces cerevisiae

2001 ◽  
Vol 152 (4) ◽  
pp. 851-856 ◽  
Author(s):  
Jill L. Johnson ◽  
Elizabeth A. Craig
Keyword(s):  
Sequence Similarity ◽  
Substrate Binding ◽  
Critical Role ◽  
Binding Domain ◽  
In Vivo Test ◽  
Protein Substrates ◽  
Unfolded Protein ◽  
Substrate Binding Domain

In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding. However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained. As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes. Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions. Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain. We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.

scholarly journals Comparison of carbohydrate ABC importers from Mycobacterium tuberculosis

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lilia I. De la Torre ◽  
José G. Vergara Meza ◽  
Sindy Cabarca ◽  
André G. Costa-Martins ◽  
Andrea Balan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Abc Transporters ◽  
Binding Proteins ◽  
Substrate Binding ◽  
Regulatory Region ◽  
Nucleotide Binding ◽  
Transmembrane Domains ◽  
Pathogenic Species ◽  
Binding Domains

Abstract Background Mycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions. Results Through the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge. Conclusions The presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.

scholarly journals Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain

2021 ◽  
Author(s):  
Marija Iljina ◽  
Hisham Mazal ◽  
Pierre Goloubinoff ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Substrate Binding Domain
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