The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site

Abstract

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25°C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37°C) and stress (42°C) temperatures, removal of the N-terminal half of ␣B and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and ␤-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.

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

“…Furthermore, binding of the substrate is directly coupled to these NBD conformational changes due to its direct stabilizing effect on the SBD lid interface and an indirect destabilizing effect on the NBD-SBD interface, clearly showing that each of these two endstates comprises distinct intrinsic interactions between two structural elements simultaneously, whereas the third state is a transition state showing interactions between all three structural elements [88]. The identification of this intricate allosteric network by NMR explained earlier observations that mutations within the SBD-lid interface led to a decrease in substrate affinity and that ATPase stimulation is coupled to substrate affinity [354][355][356].…”

DnaJ Recruits DnaK to Protein Aggregates

“…Substrate association and dissociation rate constants for DnaK2A are around 2-fold higher than those of WT DnaK, without changing the affinity for the peptide (26), in agreement with the values reported for similar full-length Hsc70 mutants (50,51). However, the increase is significantly higher (ϳ100-fold) for lidless variants of DnaK (52,53) and for the single point mutant DnaK1A (26), suggesting that interactions other than those forming the latch are more effective in controlling the accessibility of the peptide binding site. Data presented herein indicate that the latch seems to be more important in defining the affinity of DnaK for DnaJ than for peptide substrates, as previously suggested.…”

Ionic Contacts at DnaK Substrate Binding Domain Involved in the Allosteric Regulation of Lid Dynamics

“…In the presence of wt DnaK, the anisotropy value increases as the peptide binds DnaK, as previously reported (16,23,32). Equilibrium binding curves were obtained with increasing protein concentrations, and the anisotropy increase for each protein was fitted to a single site binding model (Fig.…”

“…The second mutant, DnaK2A, bears two mutations (D540A/ K548A) that break a salt bridge and a hydrogen bond between the C-terminal part of helix B and the outer loops of the ␤-subdomain. These interactions are part of the so-called "latch," that is believed to control the accessibility of the peptide binding site (1,6,13,16,30,31). The third mutant, DnaK3A, holds three substitutions (E531A/R536A/R547A) that hamper formation of two salt bridges and a hydrogen bond between residues at helix B and helices C and D of the lid.…”

“…The distal part of helix B, together with helices C, D, and E, builds up a hydrophobic helical core that acts as a "lid" (6,9). Although interdomain coupling occurs in the absence of the lid and the bound peptide contacts the ␤-structure but not the ␣-helical subdomain, its presence has been confirmed to be essential in stabilizing Hsp70-substrate complexes (14,15), especially at stress temperatures (16).…”