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

Abstract

The molecular chaperone DnaK assists protein folding and refolding, translocation across membranes, and regulation of the heat shock response. In Escherichia coli, the protein is a target for insect-derived antimicrobial peptides, pyrrhocoricins. We present here the X-ray crystallographic analysis of the E. coli DnaK substratebinding domain in complex with pyrrhocoricin-derived peptide inhibitors. The structures show that pyrrhocoricins act as site-specific, dual-mode (competitive and allosteric) inhibitors, occupying the substrate-binding tunnel and disrupting the latch between the lid and the ␤-sandwich. Our structural analysis revealed an allosteric coupling between the movements of the lid and the interdomain linker, identifying a previously unknown mechanism of the lid-mediated regulation of the chaperone cycle.DnaK is the bacterial molecular chaperone from the Hsp70 (70-kDa heat shock protein) family that assists many cellular processes involving proteins in their nonnative conformations, such as folding of newly synthesized proteins, protein translocation across membranes, refolding of misfolded and aggregated proteins, degradation of unstable proteins, and regulation of the heat shock response (for reviews, see references 10, 28, and 42). The chaperone function of DnaK is based on its ability to transiently bind to exposed stretches of hydrophobic residues in partially or fully unfolded proteins in an ATP-controlled fashion, thereby preventing aggregation and misfolding. DnaK recognizes short peptide sequences containing up to five consecutive hydrophobic residues (with leucine found frequently in the middle), flanked preferentially by basic residues (42, 43). The molecularchaperone activity is functionally linked with ATP hydrolysis; the substrate-binding and release cycle is driven by the switching between the ATP-bound state, with low affinity and a high exchange rate for substrates, and the ADP-bound state, with high affinity and a low exchange rate for substrates.In vivo, DnaK activity is supported by two cochaperones, GrpE, which facilitates the ADP/ATP exchange, and DnaJ, which stimulates ATP hydrolysis and thus aids the peptide capture (10,25,28,38,54). DnaJ itself also recognizes exposed stretches of hydrophobic residues in partially unfolded or denatured proteins, with specificity overlapping with that of DnaK (44). It is therefore thought that DnaJ serves as a scanning factor for DnaK by binding specific unfolded substrates and presenting them to the ATP-bound form of DnaK.Escherichia coli DnaK is composed of two domains: an Nterminal ATPase domain (residues 1 to 387) and a C-terminal substrate-binding domain (SBD) (residues 388 to 638) (10).The latter is made up of an 18-kDa ␤-sandwich subdomain that holds the substrate-binding cleft and a C-terminal ␣-helicalbundle "lid" subdomain that stabilizes the complex with the peptide substrate and controls the accessibility of the peptide binding site but does not interact with the substrate directly (5, 55). Removal of the lid subdomain by truncatio...

Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop

“…Docking of the atomic structures of an antibody molecule 30 and Hsp70 SBD (ref. 31) into their corresponding masses reinforced the location of the latter. This result was confirmed by an independent experiment using the Fab (Fig.…”

Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin

“…As PrAMPs bind to DnaK with only a 7-to 8-residue-long sequence stretch (9,17,20), whereas both termini protrude out of the PrAMP-DnaK complex, an influence of the penetratin sequence on the inhibition of DnaK appears unlikely. Furthermore, the intracellular distributions of PrAMPs and penetratin-PrAMP conjugates appeared to be identical.…”

“…Recently, several laboratories have started to optimize PrAMPs for clinical applications, e.g., A3-APO (25), Bac7 (12), oncocin (16), and Api88 (9), which proved successful in rodent infection models (1,15,32). Mechanistically, PrAMPs and the related designer peptides enter the bacteria and kill them, most likely by inhibiting the 70-kDa bacterial chaperone DnaK (17,20,24). The low toxicity toward mammalian cell lines is attributed to the fact that short PrAMPs appear to enter only some cells of the immune system, without inhibiting their intracellular chaperones, but are excluded from other mammalian cells (18,26,33).…”

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

“…The MIC values of these Api137 analogs indicated that the N-terminal sequence tolerated substitutions without major effects on the antibacterial activity (Fig. 4A), although apidaecins and Api88 are presumed to inhibit DnaK by binding of residues 3 to 11 to the substrate binding domain of DnaK (19,35,36). Substitutions of C-terminal residues (Pro-11 to Leu-18) significantly reduced the antibacterial activity in most cases.…”