Direct Comparison of a Stable Isolated Hsp70 Substrate-binding Domain in the Empty and Substrate-bound States

Abstract

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...

Allosteric Coupling between the Lid and Interdomain Linker in DnaK Revealed by Inhibitor Binding Studies

“…The structures of the separate ␤-sandwich and the helicallid domains, and the mode of the peptide binding, are essentially identical in all conformers, suggesting that the two subdomains of SBD move as rigid bodies. This is consistent with previous observations that, when expressed on their own, these domains form stably folded and quite rigid structures (5,49). The two extreme positions of the helical lid in the observed spectrum of conformations are defined by the structures PDB accession number 1dkx (SBD/NR) and chain B of the SBD/ short-peptide complex (Fig.…”

“…The spatial arrangement of the ATPase domain and the SBD, connected by the linker, changes significantly between the ADPand ATP-bound states. In the ADP-bound state, the two domains are in disjoined conformations and do not interact (49,50). In the ATP-bound state, the ATPase domain docks onto the SBD, inducing concerted conformational changes in both the ␤-sandwich and helical-lid subdomains of the SBD (8,33,50).…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…Extension of this study to a twodomain construct containing both NBD and SBD enabled for the first time analysis of the relative orientation of the two domains on the basis of chemical shifts as well as RDCs, highlighting a relatively rigid overall structure that shows allosteric adaptations due to nucleotide and/or substrate binding which are transmitted via subtle structural changes at the domain interface [123]. In parallel, studies on E. coli DnaK by the Gierasch lab revealed initially pH-driven conformational equilibria within the SBD, and showed by chemical shift perturbation and hydrogen/deuterium exchange that for the two-domain construct there is no interaction in the nucleotide-free state and that only nucleotides drive the allosteric adaptations [149,150].…”

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

“…Both DnaK and lidless DnaK(1-517) were dialyzed in 4 liters of buffer containing 25 mM HEPES-NaOH, pH 7, 50 mM KCl, 5 mM MgCl 2 , and 5 mM ␤-mercaptoethanol for 4 days with 3 exchanges per day to remove nucleotide. The plasmid expressing DnaK(387-552)L542Y,L543E (DnaK(387-552)-ye), with an N-terminal histidine tag, has been described previously (36). DnaK(387-552)-ye was expressed in E. coli NapIV cells and purified as previously described (36), except a step gradient up to 200 mM imidazole was used in place of a linear gradient, and protein was not purified from inclusion bodies.…”