Search

The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal of the neuron. The SNARE complex is composed of four helices contributed by three proteins: one from syntaxin (localized at the plasma membrane), one from synaptobrevin (localized at the synaptic vesicle), and two from the intrinsically disordered and aggregation-prone synaptosomal-associated 25 kDa protein (SNAP-25), which is localized to the plasma membrane by virtue of palmitoylation of cysteine residues. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its J-protein co-chaperone CSPα. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25, disfavoring its aggregation and keeping it in a folding state competent for SNARE complex formation. To test this hypothesis, we used a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. We showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25-derived peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, including native-PAGE, binding affinity by fluorescence anisotropy, ATPase-activity of Hsc70, and NMR. We have identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell to facilitate SNARE complex formation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15., Competing Interests: Competing interests statement:The authors declare no competing interest.

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hsp70 molecular chaperones play central roles in maintaining a healthy cellular proteome. Hsp70s function by binding to short peptide sequences in incompletely folded client proteins, thus preventing them from misfolding and/or aggregating, and in many cases holding them in a state that is competent for subsequent processes like translocation across membranes. There is considerable interest in predicting the sites where Hsp70s may bind their clients, as the ability to do so sheds light on the cellular functions of the chaperone. In addition, the capacity of the Hsp70 chaperone family to bind to a broad array of clients and to identify accessible sequences that enable discrimination of those that are folded from those that are not fully folded, which is essential to their cellular roles, is a fascinating puzzle in molecular recognition. In this article we discuss efforts to harness computational modeling with input from experimental data to develop a predictive understanding of the promiscuous yet selective binding of Hsp70 molecular chaperones to accessible sequences within their client proteins. We trace how an increasing understanding of the complexities of Hsp70-client interactions has led computational modeling to new underlying assumptions and design features. We describe the trend from purely data-driven analysis toward increased reliance on physics-based modeling that deeply integrates structural information and sequence-based functional data with physics-based binding energies. Notably, new experimental insights are adding to our understanding of the molecular origins of "selective promiscuity" in substrate binding by Hsp70 chaperones and challenging the underlying assumptions and design used in earlier predictive models. Taking the new experimental findings together with exciting progress in computational modeling of protein structures leads us to foresee a bright future for a predictive understanding of selective-yet-promiscuous binding exploited by Hsp70 molecular chaperones; the resulting new insights will also apply to substrate binding by other chaperones and by signaling proteins.

Dahiya et al. (2022) and Biebl et al. (2022) present mechanistic insights into the Hsp40/Hsp70/Hsp90 chaperone teamwork and the co-chaperones that participate in this network., (Copyright © 2022 Elsevier Inc. All rights reserved.)

To help cells cope with protein misfolding and aggregation, Hsp70 molecular chaperones selectively bind a variety of sequences ("selective promiscuity"). Statistical analyses from substrate-derived peptide arrays reveal that DnaK, the E. coli Hsp70, binds to sequences containing three to five branched hydrophobic residues, although otherwise the specific amino acids can vary considerably. Several high-resolution structures of the substrate -binding domain (SBD) of DnaK bound to peptides reveal a highly conserved configuration of the bound substrate and further suggest that the substrate-binding cleft consists of five largely independent sites for interaction with five consecutive substrate residues. Importantly, both substrate backbone orientations (N- to C- and C- to N-) allow essentially the same backbone hydrogen-bonding and side-chain interactions with the chaperone. In order to rationalize these observations, we performed atomistic molecular dynamics simulations to sample the interactions of all 20 amino acid side chains in each of the five sites of the chaperone in the context of the conserved substrate backbone configurations. The resulting interaction energetics provide the basis set for deriving a predictive model that we call Paladin (Physics-based model of DnaK-Substrate Binding). Trained using available peptide array data, Paladin can distinguish binders and nonbinders of DnaK with accuracy comparable to existing predictors and further predicts the detailed configuration of the bound sequence. Tested using existing DnaK-peptide structures, Paladin correctly predicted the binding register in 10 out of 13 substrate sequences that bind in the N- to C- orientation, and the binding orientation in 16 out of 22 sequences. The physical basis of the Paladin model provides insight into the origins of how Hsp70s bind substrates with a balance of selectivity and promiscuity. The approach described here can be extended to other Hsp70s where extensive peptide array data is not available., Competing Interests: The authors have declared that no competing interests exist.

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms., Competing Interests: The authors declare no competing interest.

The Hsp70 family of chaperones works with its co-chaperones, the nucleotide exchange factors and J-domain proteins, to facilitate a multitude of cellular functions. Central players in protein homeostasis, these jacks-of-many-trades are utilized in a variety of ways because of their ability to bind with selective promiscuity to regions of their client proteins that are exposed when the client is unfolded, either fully or partially, or visits a conformational state that exposes the binding region in a regulated manner. The key to Hsp70 functions is that their substrate binding is transient and allosterically cycles in a nucleotide-dependent fashion between high- and low-affinity states. In the past few years, structural insights into the molecular mechanism of this allosterically regulated binding have emerged and provided deep insight into the deceptively simple Hsp70 molecular machine that is so widely harnessed by nature for diverse cellular functions. In this review, these structural insights are discussed to give a picture of the current understanding of how Hsp70 chaperones work., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The 70-kDa heat shock proteins (Hsp70s) are molecular chaperones that perform a wide range of critical cellular functions. They assist in the folding of newly synthesized proteins, facilitate assembly of specific protein complexes, shepherd proteins across membranes, and prevent protein misfolding and aggregation. Hsp70s perform these functions by a conserved mechanism that relies on allosteric cycles of nucleotide-modulated binding and release of client proteins. Current models for Hsp70 allostery have come from extensive study of the bacterial Hsp70, DnaK. Extending our understanding to eukaryotic Hsp70s is extremely important not only in providing a likely common mechanistic framework but also because of their central roles in cellular physiology. In this study, we examined the allosteric behaviors of the eukaryotic cytoplasmic Hsp70s, HspA1 and Hsc70, and found significant differences from that of DnaK. We found that HspA1 and Hsc70 favor a state in which the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) are intimately docked significantly more as compared to DnaK. Past work established that the NBD-SBD interface and the helical lid-β-SBD interface govern the allosteric landscape of DnaK. Here, we identified sites on these interfaces that differ between eukaryotic cytoplasmic Hsp70s and DnaK. Our mutational analysis has revealed key evolutionary variations that account for the population shifts between the docked and undocked conformations. These results underline the tunability of Hsp70 functions by modulation of allosteric interfaces through evolutionary diversification and also suggest sites where the binding of small-molecule modulators could influence Hsp70 function., Competing Interests: The authors declare no conflict of interest.

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains ( viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The allosteric mechanism of Hsp70 molecular chaperones enables ATP binding to the N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to the C-terminal substrate-binding domain (SBD) and substrate binding to enhance ATP hydrolysis. Cycling between ATP-bound and ADP/substrate-bound states requires Hsp70s to visit a state with high ATPase activity and fast on/off kinetics of substrate binding. We have trapped this "allosterically active" state for the E. coli Hsp70, DnaK, and identified how interactions among the NBD, the β subdomain of the SBD, the SBD α-helical lid, and the conserved hydrophobic interdomain linker enable allosteric signal transmission between ligand-binding sites. Allostery in Hsp70s results from an energetic tug-of-war between domain conformations and formation of two orthogonal interfaces: between the NBD and SBD, and between the helical lid and the β subdomain of the SBD. The resulting energetic tension underlies Hsp70 functional properties and enables them to be modulated by ligands and cochaperones and "tuned" through evolution., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators., (© 2011 American Chemical Society)

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including choice of site for ligation with retention of function, optimization of ligation yield, and purification from unreacted domains. Ligated semilabeled DnaK was successfully produced with a Cys residue at position 383, and the ligated product harboring the Cys mutation was confirmed to be functional and identical to an expressed Cys-containing two-domain construct. The NMR spectrum of the segmentally labeled protein was considerably simplified, enabling unequivocal assignment and enhanced analysis of dynamics, as a prelude to exploring the energy landscape for allostery in the Hsp70 family., (2010 Wiley Periodicals, Inc.)

Molecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP. These data suggest a relatively simple circadian system in which a single oscillator provides temporal information for all downstream processes. However, in vivo the situation is more complex, and additional components contribute to the maintenance of a normal period, the resetting of relative phases of circadian oscillations, and the control of rhythms of gene expression. We show here that two well-studied promoters in the S. elongatus genome report different circadian periods of expression under a given set of conditions in wild-type as well as mutant genetic backgrounds. Moreover, the period differs between these promoters with respect to modulation by light intensity, growth phase, and the presence or absence of a promoter-recognition subunit of RNA polymerase. These data contrast sharply with the current clock model in which a single Kai-based oscillator governs circadian period. Overall, these findings suggest that complex interactions among the circadian oscillator, perhaps other oscillators, and other cellular machinery result in a clock that is plastic and sensitive to the environment and to the physiological state of the cell.

The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying prokaryotic circadian rhythms. Although S. elongatus does not display an easily measurable overt circadian behavior, its gene expression is under circadian control; hence, a "behavior" is created by linking a cyanobacterial promoter to either the bacterial luxAB or firefly luc luciferase genes to create reporter fusions whose activity can be easily monitored by bioluminescence. Numerous vectors have been created in our lab for introducing luciferase reporter genes into the S. elongatus chromosome. A choice of methods and equipment to detect light production from the luciferase fusions provides a means for high-throughput, automated mutant screens as well as testing rhythms from two promoter fusions within the same cell culture.

Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.