d-Peptide Ligands for the Co-chaperone DnaJ

Abstract

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudopeptides of D-amino Chaperones of the Hsp70 1 family fulfill essential functions in protein folding by preventing and reversing off-pathway interactions that lead to aggregation (1, 2). Hsp70s are also required for membrane translocation of precursor polypeptides (3, 4) and participate in the degradation of misfolded proteins (2).The Hsp70 chaperone system of Escherichia coli comprises the Hsp70 homolog DnaK and the co-chaperones DnaJ and GrpE. DnaK consists of a 44-kDa ATPase domain, the crystal structure of which has recently been determined in a complex with a dimer of truncated GrpE (5), and a 27-kDa peptidebinding domain, the crystal structure of which has been solved in a complex with a synthetic peptide ligand (6). The chaperone effects of the DnaK/DnaJ/GrpE system are based on the ability of DnaK to bind extended hydrophobic segments of proteins in a reversible manner and possibly to exert conformational work on them (2, 7). Binding and release of polypeptides are modulated by ATP binding and hydrolysis (8), which on their part are controlled by the co-chaperones GrpE and DnaJ, respectively (7, 9). DnaJ interacts with DnaK through its highly conserved N-terminal J-domain (10, 11). However, DnaJ appears to exert also a chaperone function of its own (12). The co-chaperone associates with denatured polypeptides independently of DnaK, e.g. luciferase and rhodanese (13, 14), preventing their aggregation and targeting them to DnaK.In previous studies, we have found various peptides derived from the presequence of mitochondrial aspartate aminotransferase to be high affinity ligands of DnaK (7).2 In a search for peptide mimetics that might be used as inhibitors of the chaperone system, we tested normal all-D and retro all-D (retroinverso) analogs of a parent all-L peptide for binding to DnaK and DnaJ. In the normal all-D peptide mimetics, all L-amino acid residues have been replaced by the corresponding D-enantiomers; the main chains of these peptide analogs have the same direction as in the parent peptides, whereas the side chains occupy enantiomorphic positions. In contrast, retro all-D peptides, i.e. mimetic peptides of D-amino acids with, as compared with the reference L-peptides, reverse sequence, exhibit in extended conformation the same side chain topology as their native counterparts (15). In certain cases they may therefore retain the biological activity of the reference L-peptides while being much less susceptible to proteolytic degradation (16 -18).Here we show that the co-chaperone DnaJ binds the tested pseudo-peptides with similar affinities as the parent all-L peptide. In contrast, DnaK did not bind any of the D-peptides with measurable affinity. EXPERIMENTAL PROCEDURESPurification of Proteins-DnaK from an overproducing strain of E. coli bearin...

The insect antimicrobial peptide, -pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

“…For example, protamine, which is a polycationic peptide with antimicrobial activity, binds to DnaK in a manner similar to short synthetic peptides [22]. (iii) The inactivity of D D -PYR to DnaK agrees with previous findings that DnaK does not bind peptides composed of all-D D amino acids [5]. Overall, our results are consistent with L L -PYR being a pseudo-substrate of DnaK, i.e., a competitive inhibitor.…”

“…Because the DnaK chaperone machine is absolutely essential to repair partially denatured proteins that occur as a consequence of stress, inhibiting DnaK would devastate a bacterial cell. There have been intriguing reports of novel classes of DnaK (Hsp70) [4] and DnaJ (Hsp40) [5] inhibitors. Such inhibitors, if they selectively bind to the prokaryotic but not to the eukaryotic chaperone, could be an important new class of antimicrobial agents.…”

d-Peptides as Inhibitors of the DnaK/DnaJ/GrpE Chaperone System

“…The radioactive ADP product was separated from radioactive ATP by thin layer chromatography, and the radioactivity of ADP and ATP was determined with a liquid scintillation counter. The binding rate constants of DnaJ for acrylodan-labeled peptides were determined with a Spex Fluorolog spectrofluorimeter as described (7). The excitation wavelength was set at 370 nm (band pass, 4.6 nm) with the emission wavelength set at 520 nm (band pass, 4.6 nm).…”

“…DnaJ also exerts a chaperone action on its own; upon association with denatured polypeptides, such as luciferase or rhodanese, it may prevent their aggregation (3,6). Recently, it has been shown that D-peptides bind to DnaJ but not to DnaK (7,8). D-Peptides bind to the same site of DnaJ as L-peptides (7).…”

“…Recently, it has been shown that D-peptides bind to DnaJ but not to DnaK (7,8). D-Peptides bind to the same site of DnaJ as L-peptides (7). Here we report that two retro-all D-peptides derived from the NH 2 -terminal segment of rhodanese inhibit the DnaK/DnaJ/GrpE chaperone system in refolding denatured firefly luciferase.…”