The bacterial protein DnaK promotes folding of newly synthesized
polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted
refolding is especially important under stress conditions induced by antibiotic
therapies reducing the desired bactericidal effects. DnaK is supposedly targeted
by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli
ΔdnaK mutants and wild type strains are equally susceptible indicating further
intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK-
complexes revealed forward and reverse binding modes at the substrate
binding domain. Here, we used these ligand-target structures for the first time to
rationally optimize peptides using molecular modeling and docking leading to the
prediction of four-residue long sequences for improved binding to DnaK. When these sequences
were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced
dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the
X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to
stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound
to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar
range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations
against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants.
However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA
transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to
speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.
Keywords: Apidaecin, drug design, oncocin, fluorescence polarization, molecular docking, molecular dynamics interactions.
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