Rational Design of Novel Peptidic DnaK Ligands

Abstract

The hsp70 chaperone DnaK from E. coli plays a major role in cellular stress response and is involved in assisted protein folding in vivo. By screening a combinatorial peptide library, we identified several DnaK‐specific peptide ligands with nanomolar affinities, which are able to inhibit the secondary amide peptide bond cis/trans isomerase (APIase) activity of DnaK, as well as DnaK/DnaJ/GrpE‐assisted refolding of firefly luciferase. Our designed DnaK inhibitors have the capability to penetrate E. coli cells and feature a high protease resistance. Once inside the cell, they physically target DnaK. NMR‐based 1H/15N‐HSQC experiments furthermore confirmed that the designed peptidic ligands all bind in an identical manner to the conventional peptide‐binding site of DnaK. The subsequent blocking of DnaK function apparently results in the observed antibacterial effects on E. coli cells, with minimum inhibitory concentrations in the range of 100 μM.

Bactericidal oncocin derivatives with superior serum stabilities

“…In light of these facts, several laboratories have recently designed and optimised novel proline-rich AMP analogues with interesting antibacterial activities and with no toxic effects on mammalian cell lines or erythrocytes [7][8][9]. The high proline content also provides fairly good stability against proteolytic degradation in mammalian serum, which can be even further reduced by stabilising the cleavage sites.…”

Rational Design of Oncocin Derivatives with Superior Protease Stabilities and Antibacterial Activities Based on the High‐Resolution Structure of the Oncocin‐DnaK Complex

“…These values correspond to binding constants reported for other DnaK-binding sequences, ranging from 0.1 to 10 mm. [14] Cocrystallization of oncocin O2 with the substrate binding domain of DnaK (residues 389 to 607), demonstrated that oncocin residues 4 to 10 (PPYLPR) bound to the peptide binding site of DnaK, whereas the remaining terminal residues of the peptide were flexible (Figures 1 and S4-S8). [15] Interestingly, this sequence stretch matched the residues identified by the Ala-scan as crucial for antibacterial activity.…”

“…Oncocin was digested between 15 R and 16 I (Figure 2). The two shorter peptides ( [1][2][3][4][5][6][7][8][9][10][11][12][13] O2 and [1][2][3][4][5][6][7][8][9][10] O2) with a C-terminal Pro were probably the results of degradation of [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] O2 by exoproteases. Peptide O2 was slightly more stable (t1 = 2 = 125 min), but also cleaved before Ile16.…”

“…[c] This halflife was estimated from the MALDI signal intensities for peptide O5 and its metabolite [1][2][3][4][5][6][7][8][9][10][11][12][13][14] O5, because they both co-eluted on RP-HPLC.…”

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching

“…Other factors which are essential for an efficient use of an AMP are its membrane penetration, serum stability, and half-life in an organism. All of these factors can be influenced by slight modifications in the amino acid sequences of AMPs [6], and relatively subtle amino acid substitutions have been shown to dramatically improve the antimicrobial activity of oncocin derivatives in certain cases [7,8]. However, the redesign of a peptide of, e.g., 20 amino acids in length, is a complex and timeconsuming task as theoretically every amino acid position can be replaced with every existing naturally occurring and synthetic amino acid.…”