Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.

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Proteins mediate transmission of signals along intercellular and intracellular pathways and between the exterior and the interior of a cell. The dynamic properties of signaling proteins are crucial to their functions. We discuss emerging paradigms for the role of protein dynamics in signaling. A central tenet is that proteins fluctuate among many states on evolutionarily selected energy landscapes. Upstream signals remodel this landscape, causing signaling proteins to transmit information to downstream partners. New methods provide insight into the dynamic properties of signaling proteins at the atomic scale. The next stages in the signaling hierarchyhow multiple signals are integrated and how cellular signaling pathways are organized in space and time-present exciting challenges for the future, requiring bold multidisciplinary approaches.Transmission of signals between cells, within cells, and from the extracellular environment to the cellular interior is essential to life. In recent years, we have gained tremendous knowledge of the interacting networks that act as communication pathways for cellular signaling, culminating with extensive maps of "interactomes" based on genetic and physical interaction data [e.g., (1)]. Yet we know far less about how signals are passed from one component of a network to another. This puzzle can be viewed at the level of the protein machines that make up signaling networks: How does information, generally mediated by a binding interaction or a covalent modification, get relayed to the downstream member of the pathway?It is increasingly apparent that signaling relies on the intrinsic dynamic properties of proteins and that proteins relay signals by shifting among different fluctuating energy states in response to one or more inputs. This emerging view of signaling raises many compelling questions: What is the genetic information that encodes the functionally productive dynamic properties of a protein? How do individual protein domains cooperate to form signaling pathways? How are signals integrated in multicomponent interconnecting networks? How do the dynamics of signaling proteins ultimately determine the response times of cells to signals and the time scales for signal propagation? We are beginning to develop the methods and principles to address these questions, but many challenges lie ahead. Our ability to develop therapeutic modulators of signaling and to reengineer cellular communication pathways will rely on progress in this fascinating but complex arena.The intrinsic motions of proteins are determined by the covalent and noncovalent restraining forces that hold them together. The result is a symphony of dynamic modes oscillating at frequencies from picoseconds to milliseconds or even seconds. Tweaking a protein by a binding interaction or chemical modification alters this symphony, either gently changing its pitch or abruptly shifting the collective harmony. Just as the ability of a protein to fold is now understood to be best described by an "energy landscape," w...

The GroES heptamer forms a dome, approximately 75 A in diameter and 30 A high, with an 8 A orifice in the centre of its roof. The 'mobile loop' segment, previously identified as a GroEL binding determinant, is disordered in the crystal structure in six subunits; the single well-ordered copy extends from the bottom outer rim of the GroES dome, suggesting that the cavity within the dome is continuous with the polypeptide binding chamber of GroEL in the chaperonin complex.

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.

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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.