The molecular chaperone heat shock protein 70 (Hsp70) plays a vital role in cellular processes, including protein folding and assembly, and helps prevent aggregation under physiological and stressrelated conditions. Although the structural changes undergone by full-length client proteins upon interaction with DnaK (i.e., Escherichia coli Hsp70) are fundamental to understand chaperone-mediated protein folding, these changes are still largely unexplored. Here, we show that multiple conformations of the SRC homology 3 domain (SH3) client protein interact with the ADP-bound form of the DnaK chaperone. Chaperone-bound SH3 is largely unstructured yet distinct from the unfolded state in the absence of DnaK. The bound client protein shares a highly flexible N terminus and multiple slowly interconverting conformations in different parts of the sequence. In all, there is significant structural and dynamical heterogeneity in the DnaK-bound client protein, revealing that proteins may undergo some conformational sampling while chaperonebound. This result is important because it shows that the surface of the Hsp70 chaperone provides an aggregation-free environment able to support part of the search for the native state. However, ribosome-bound, newly synthesized, stress-unfolded, large or highly concentrated proteins often cannot fold efficiently in vitro and/or are particularly prone to aggregation. Remarkably, in the cellular environment, molecular chaperones and the ribosome help ensure that all proteins attain their native state (2-5). Molecular chaperones are overexpressed under stressrelated conditions and help maintain a population of correctly folded proteins. Some chaperones also interact with cellular proteins cotranslationally and posttranslationally under nonstress conditions (6).The heat shock protein 70 (Hsp70; ∼70 kDa) class of molecular chaperones (7) are single-chain proteins comprising a nucleotidebinding domain (NBD; ca. 42 kDa) capable of hydrolyzing ATP, a substrate-binding domain (SBD; ca. 27 kDa) devoted to interacting with the substrate, and a flexible linker connecting the two domains (6, 8) ( Fig. 1 A-C). Hsp70 is involved in a variety of protein folding and trafficking processes (9), and it plays major yet diverse roles in disease. For instance, overexpression of Hsp70 is related to cancer (10), and Hsp70 overexpression in transgenic mice leads to spontaneous development of T-cell lymphoma (10, 11). In addition, Hsp70 plays a protective role against a number of neurodegenerative diseases by preventing protein aggregation (12)(13)(14).Due to the central importance of Hsp70, significant efforts have been devoted to understand its function, including how ATP hydrolysis modulates substrate affinity, how this chaperone interacts with its cochaperones, and how Hsp70 undergoes allosteric regulation during the different stages of its functional cycle (8,15).On the other hand, a crucial piece of information for understanding the role of Hsp70 in the mechanism of protein folding (i.e., how the conformation of...

Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

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