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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The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains.

The Hsp70 family of molecular chaperones acts to prevent protein misfolding, import proteins into organelles, unravel protein aggregates, and enhance cell survival under stress conditions. These activities are all mediated by recognition of diverse hydrophobic sequences via a C-terminal substrate-binding domain. ATP-binding/hydrolysis by the N-terminal ATPase domain regulates the interconversion of the substrate-binding domain between low and high affinity conformations. The empty state of the substrate-binding domain has been difficult to study because of its propensity to bind nearly any available protein chain, even if only modestly hydrophobic. We have generated a new stable construct of the substrate-binding domain from the Escherichia coli Hsp70, DnaK, which has enabled us to compare the empty and peptide-bound conformations using NMR chemical shift analysis and hydrogen-deuterium exchange. We have determined that the empty state is, overall, quite similar to the peptide-bound state, contrary to a previous report. Peptide binding leads to a subtle alteration in the packing of the ␣-helical lid relative to the ␤-subdomain. Significantly, we have shown that the chemical shifts of the substrate-binding domain and the ATPase domain do not change when they are placed together in a two-domain construct, whether or not peptide is bound, suggesting that, in the absence of nucleotide, the two domains of E. coli DnaK do not interact. We conclude that the isolated substrate-binding domain exists in a stable high affinity state in the absence of influence from a nucleotide-bound ATPase domain.Escherichia coli DnaK is a member of the Hsp70 family of molecular chaperones, which facilitate folding of nascent protein chains, enhance recovery after cellular stresses such as heat shock, aid in protein translocation across membranes, and participate in protein disaggregation and degradation (1). In addition to these very general functions, which rely on ATP-regulated binding to short hydrophobic stretches of substrate proteins, Hsp70s also play specific cellular roles in disrupting macromolecular complexes such as clathrin coats. When ADP is bound to the 45-kDa N-terminal ATPase domain, the 25-kDa C-terminal substrate-binding domain (SBD) 3 binds substrates with high affinity and slow on/off kinetics. However, ATP binding converts the SBD to a low affinity conformation characterized by rapid substrate on/off rates and enhanced proteolytic susceptibility (2, 3). Small angle x-ray scattering data collected on the related protein Hsc70 (4) implicates a more intimate association between the two domains in the ATP-bound conformation, and in this state, the sole intrinsic tryptophan of DnaK, Trp-102, is buried at the interface with the SBD (3, 5). Not only does nucleotide binding influence the peptide-binding pocket, but peptide binding also speeds the ATP hydrolysis rate, which is the rate-limiting step of the cycle for E. coli DnaK. The lack of a high resolution structure including both domains has hampered our understanding o...

SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564 -579) and to the extreme C-terminal segment of SecA (residues 864 -901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA.