Untitled

Abstract

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions

“…Upon ATP binding there is movement of loops L2,3, L4,5, and L6,7 into positions that engage the NBD [16]. These rearrangements of the β strands dictate how ATP allosterically couples the SBD and NBD, thereby regulating substrate binding and triggering ATP hydrolysis [71]. Not surprisingly, mutations in residues within this region disrupt SBD-NBD coupling [72–74].…”

Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

“…However, these results do not distinguish whether the lid domain is required for wild-type function of DnaK by enabling chaperone activity or by determining DnaK protein stability in the cell. Biochemical characterization of lidless E. coli DnaK indicates that the lid promotes activity by enhancing substrate binding (32,33). To test whether removal of the C-terminal lid domain affects DnaK stability, we evaluated steady-state levels of each DnaK allele by immunoblotting with anti-DnaK antiserum (Fig.…”

“…These results are consistent with studies of E. coli demonstrating that lid-less DnaK retains functionality, albeit at a reduced level. Specifically, C-terminally truncated DnaK maintains ATP-dependent binding of peptide substrates, undergoes substrate-activated ATP-hydrolysis in vitro, and supports replication of bacteriophage in vivo (32,33). While the lid domain is not necessary for substrate binding, it does serve to enhance affinity for substrate and increase the lifetime of DnaK-substrate complexes.…”

“…1D, and the affinity for substrate is dramatically enhanced (29)(30)(31). ATP-dependent regulation of substrate affinity is independent of the C-terminal helical lid, although this domain enhances the residence time of substrates on DnaK, which reduces protein aggregation and facilitates substrate folding (32,33).…”

HSPA8/HSC70 chaperone protein

“…This closure of the lid enables the chaperone to hold onto the peptide very efficiently, and deletion of the lid results in a decrease of peptide affinity by 5-fold, as well as an increase of the ATP-dependent peptide release by 2 orders of magnitude. 69 It is worth noting that most of the experiments done regarding the binding mechanism of proteins of the HSP70 family have been realized with peptides, whereas the main in vivo substrates for the chaperones could also be partially unfolded proteins or even native-state ones. Therefore, the necessary closing of the lid to ensure correct binding seems puzzling since a protein would have to be mainly unfolded to be able to present short hydrophobic stretches to the SBD in the same orientation that was observed in the crystal structure.…”