Summary 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 between 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 SBD). The resulting energetic tension underlies Hsp70 functional properties and enables them to be modulated by ligands and co-chaperones and ‘tuned’ through evolution.

Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods are needed that preserve these interactions and provide information about them. In-cell NMR spectroscopy is an attractive method to study a protein’s behavior in cells because it may provide residue-level structural and dynamic information. Yet several factors limit the feasibility of protein NMR spectroscopy in cells, and among them slow rotational diffusion has emerged as the most important. In this paper, we seek to elucidate the causes of the dramatically slow protein tumbling in cells and in so doing to gain insight into how the intracellular viscosity and weak, transient interactions modulate protein mobility. To address these questions, we characterized the rotational diffusion of three model globular proteins in E. coli cells using 2D heteronuclear NMR spectroscopy. These proteins have a similar molecular size and globular fold, but very different surface properties, and indeed, they show very different rotational diffusion in the E. coli intracellular environment. Our data are consistent with an intracellular viscosity approximately eight times that of water—too low to be a limiting factor to observing small globular proteins by in-cell NMR spectroscopy. Thus, we conclude that transient interactions with cytoplasmic components significantly and differentially affect the mobility of proteins and therefore their NMR detectability. Moreover, we suggest that an intricate interplay of total protein charge and hydrophobic interactions plays a key role in regulating these weak intermolecular interactions in cells.

The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues 389 VLLL 392 ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA β-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker. T he 70-kDa heat shock proteins (Hsp70s) compose one of the most well studied and ubiquitously distributed families of allosteric proteins (1). Hsp70s assist in an extraordinarily broad spectrum of cellular processes, including protein folding, disaggregation, and translocation. All chaperone activities of Hsp70s are based on their ability to interact with short hydrophobic peptide segments of protein substrate in an ATP-dependent fashion. Hsp70s contain two domains-a 44-kDa N-terminal nucleotidebinding domain (NBD) and a 15-kDa C-terminal substrate-binding domain (SBD)-connected by a highly conserved hydrophobic linker. The allosteric cycle of Hsp70s involves an alternation between the ATP-bound state with low affinity and fast exchange rates for substrates, and the ADP-bound state with high affinity and low exchange rates for substrates. In turn, substrate binding to the SBD results in about 10-fold stimulation of ATPase activity of the NBD. However, the same ATPase stimulation can be achieved for the isolated NBD in the presence of the conserved interdomain linker sequence motif ( 389 VLLL 392 ) (2-4), indicating that the linker plays a key role in NBD function and allostery.The Hsp70 NBD belongs to the Actin/Hexokinase/Hsp70 superfamily, members of which share a number of common features (5, 6). The NBD is composed of two lobes, I and II; each lobe consists, in turn, of two subdomains: IA and IB for lobe I, and IIA and IIB for lobe II. Nucleotide binds at the bottom of the deep central cleft at the interface between subdomains IB and IIB, and all four subdomains are involved in nucleotide coordination...

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. molecular chaperone | protein quality control | NMR chemical shift perturbations | conformational selection | entropically driven allostery H eat shock protein 70 (Hsp70) molecular chaperones are central players in protein quality control systems for organisms from bacteria to humans (1, 2). All Hsp70s consist of two highly conserved domains: an N-terminal nucleotide-binding domain (NBD), which regulates the affinity of substrate binding, and a C-terminal substrate-binding domain (SBD), which binds to exposed hydrophobic stretches of client proteins and is made up of a β-sandwich domain (the βSBD), and an α-helical lid (the αLid) (Fig. 1A). Hsp70 functions rely on interdomain allostery: ATP hydrolysis in the NBD controls thermodynamics and kinetics of substrate binding and release; in turn, substrate binding to the SBD stimulates ATPase activity in the NBD (3-5).Mechanistic understanding of Hsp70 function has been greatly enhanced by recent breakthrough achievements in structural characterization of individual functional steps of the allosteric cycle for the Escherichia coli Hsp70 DnaK. New crystal structures and NMR analysis of DnaK have provided descriptions of three major functional states: ADP-bound (6, 7), 9), and ATP/substrate-bound (10), which have distinct arrangements of the four structural units NBD, βSBD, αLid, and interdomain linker (Fig. 1A). In the ADP-bound (domain-undocked, linker-unbound) state, the two DnaK domains, the NBD and SBD, behave independently, with the interdomain linker exposed to the solvent and ...

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor ζ subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.

Approach Summary Licensing status Publication and contact information Instrumentation High-speed NMR spectroscopy A combination of alternative sampling and data analysis processes enables faster completion of NMR spectroscopy experiments, thus facilitating structural genomics research. The strategy combines three data-acquisition strategies-nonlinear sampling, targeted acquisition and hyperdimensional spectroscopy-with multidimensional decomposition, a mathematical technique that had been applied to other data analysis problems but not to NMR. The 8 kDa globular protein ubiquitin and the 13 kDa naturally disordered protein ζ cyt were characterized in 0.4 and 5.2 hours, respectively, representing time savings of ∼100-fold over conventional methods. Further validation might be achieved with NMR spectroscopy experiments on known proteins in a blinded fashion. Not applicable Jaravine, V. et al.

It is well-known that structures of globular proteins in liquid and in crystalline solid are essentially identical. Many lines of evidence suggest that internal dynamics are also similar (assuming that the solid sample is well-hydrated and the measurements are conducted at the same temperature). On the basis of this premise, we undertake a combined analysis of solid- and liquid-state 15N relaxation data from a small globular protein, α-spectrin SH3 domain. The interpretation using the extended Lipari−Szabo model demonstrates that liquid R 1, R 2, NOE, and solid R 1 data measured at multiple fields are mutually consistent. To validate these results, we prepared a series of samples where the protein is dissolved in a water−glycerol solvent. The presence of glycerol ensures that the overall protein tumbling is slowed, thus increasing the visibility of nanosecond time-scale internal motions. When additional data are included in the fitting procedure, a credible picture of protein dynamics is obtained. In particular, the analysis suggests that ns time-scale motions with very low amplitude, S 2≈ 0.95, are present throughout the protein. It is envisaged that combined analyses of liquid- and solid-state data can provide an efficient method for detailed characterization of internal dynamics in proteins at multiple time scales.

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