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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.

Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, are often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7; hepatocytes; Chinese hamster ovarian cells (CHO), a common host to produce biologics; and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N- to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α 1 -antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More β -rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone β -strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization., Competing Interests: Conflicts of Interest: The authors declare that they have no conflicts of interest with the contents of this article.

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.

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

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.

Stunning advances have been achieved in addressing the protein folding problem, providing deeper understanding of the mechanisms by which proteins navigate energy landscapes to reach their native states and enabling powerful algorithms to connect sequence to structure. However, the realities of the in vivo protein folding problem remain a challenge to reckon with. Here, we discuss the concept of the "proteome folding problem"-the problem of how organisms build and maintain a functional proteome-by admitting that folding energy landscapes are characterized by many misfolded states and that cells must deploy a network of chaperones and degradation enzymes to minimize deleterious impacts of these off-pathway species. The resulting proteostasis network is an inextricable part of in vivo protein folding and must be understood in detail if we are to solve the proteome folding problem. We discuss how the development of computational models for the proteostasis network's actions and the relationship to the biophysical properties of the proteome has begun to offer new insights and capabilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This collection of articles celebrates the 50th anniversary of the Protein Data Bank (PDB), the single global digital archive of biological macromolecular structures. The impact of the PDB is immense; we have invited a number of top researchers in structural biology to illustrate its influence on an array of scientific fields. What emerges is a compelling picture of the synergism between the PDB and the explosive progress witnessed in many scientific areas. Availability of reliable, openly accessible, well-archived structural information has arguably had more impact on cell and molecular biology than even some of the enabling technologies such as PCR. We have seen the science move from a time when structural biologists contributed the lion's share of the structures to the PDB and for discussion within their community to a time when any effort to achieve in-depth understanding of a biochemical or cell biological question demands an interdisciplinary approach built atop structural underpinnings., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In part 1 of this remarkable collection, we told you the story of The Protein Data Bank (PDB) (1), which was founded 50 years ago, and we illustrated the breadth of the science contained within it with ten informative review articles. The second half of this collection is a continuation of our celebrations to mark this momentous anniversary. Part 2 provides eight more superb articles describing how the PDB has influenced biology over the course of the last half-century and how biology has fueled the deposition of impactful structures in the PDB. Here are some brief synopses of the articles you will enjoy in part 2!, Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed N -glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has N -glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway., (© 2019 Adams et al.)

The effect of mutations in individual proteins on protein homeostasis, or "proteostasis," can in principle depend on the mutations' effects on the thermodynamics or kinetics of folding, or both. Here, we explore this issue using a computational model of in vivo protein folding that we call FoldEcoSlim. Our model predicts that kinetic versus thermodynamic control of mutational effects on proteostasis hinges on the relationship between how fast a protein's folding reaction reaches equilibrium and a critical time scale that characterizes the lifetime of a protein in its environment: for rapidly dividing bacteria, this time scale is that of cell division; for proteins that are produced in heterologous expression systems, this time scale is the amount of time before the protein is harvested; for proteins that are synthesized in and then exported from the eukaryotic endoplasmic reticulum, this time scale is that of protein secretion, and so forth. This prediction was validated experimentally by examining the expression yields of the wild type and several destabilized mutants of a model protein, the mouse ortholog of cellular retinoic acid-binding protein 1., (© 2019 The Protein Society.)

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.)

Hsp70 chaperones are central hubs of the protein quality control network and collaborate with co-chaperones having a J-domain (an ∼70-residue-long helical hairpin with a flexible loop and a conserved His-Pro-Asp motif required for ATP hydrolysis by Hsp70s) and also with nucleotide exchange factors to facilitate many protein-folding processes that (re)establish protein homeostasis. The Hsp70s are highly dynamic nanomachines that modulate the conformation of their substrate polypeptides by transiently binding to short, mostly hydrophobic stretches. This interaction is regulated by an intricate allosteric mechanism. The J-domain co-chaperones target Hsp70 to their polypeptide substrates, and the nucleotide exchange factors regulate the lifetime of the Hsp70-substrate complexes. Significant advances in recent years are beginning to unravel the molecular mechanism of this chaperone machine and how they treat their substrate proteins., (© 2019 Mayer and Gierasch.)

The folding of predominantly β-sheet proteins is complicated by the presence of a large number of non-local interactions in their native states, which increase the ruggedness of their folding energy landscapes. However, forming non-local contacts early in folding or even in the unfolded state can smooth the energy landscape and facilitate productive folding. We report that several sequence regions of a β-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), populate native-like secondary structure to a significant extent in the denatured state in 8 M urea. In addition, we provide evidence for both local and non-local interactions in the denatured state of CRABP1. NMR chemical shift perturbations (CSPs) under denaturing conditions upon substitution of single residues by mutation support the presence of several non-local interactions in topologically key sites, arguing that the denatured state is conformationally restricted and contains topological information for the native fold. Among the most striking non-local interactions are those between the N- and C-terminal regions, which are involved in closure of the native β-barrel. In addition, CSPs support the presence of two features in the denatured state: a major hydrophobic cluster involving residues from various parts of the sequence and a native-like interaction similar to one identified in previous studies as forming early in folding (Budyak et al., Structure 21, 476 [2013]). Taken together, our data support a model in which transient structures involving nonlocal interactions prime early folding interactions in CRABP1, determine its barrel topology, and may protect this predominantly β-sheet protein against aggregation., (© 2018 The Protein 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.

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.)