Proline-rich Antimicrobial Peptides Optimized for Binding to Escherichia coli Chaperone DnaK

Abstract

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.

Identification and elucidation of proline‐rich antimicrobial peptides with enhanced potency and delivery

“…It will be informative to investigate the effect of C‐terminal amidation for PrAMPs on membrane transportation in future work. In addition, comparing the binding and internalization rate, oncocin shows a higher transportation rate in the presence of DnaK, which is in line with the speculation of a transportation function of DnaK for PrAMPs in the previous study (Knappe et al, ).…”

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

“…These proteases can also degrade therapeutic peptides that commonly contain basic residues important for binding to the target via ionic interactions [14,15] or cell entry in eukaryotic and prokaryotic cells [16–18]. …”

Systematic mutagenesis of oncocin reveals enhanced activity and insights into the mechanisms of antimicrobial activity

“…19 Moreover, computer simulations have been applied to rationally design stronger binding peptides to DnaK protein by substituting seven residues from the N-terminal region with four optimized residues selected from molecular modeling and docking. 21 Although six derivatives were found to bind more strongly to DnaK, the minimum inhibitory concentrations (MICs) against wild type E. coli were not improved. The use of array synthesis has been applied to optimize oncocin activity against Gram-positive Staphylococcus aureus and Pseudomonas aeruginosa, against which oncocin has rather weak activity.…”