Abstract
The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gram-negative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 µg/mL to as low as 16 µg/mL. Only a single analog exhibited improved activity over the original value of 8 µg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.
Highlights
Proline-rich antimicrobial peptides (PrAMP) have been intensively studied with special emphasis on their mode of action, protease stability, and toxicity (Scocchi et al, 2011; Hansen et al, 2012; Li et al, 2014; Böttger et al, 2017)
This study describes the influence of PrAMP ARV-1502 and 182 substituted analogs on the chaperone system of the Gramnegative bacterium E. coli and the Gram-positive bacterium S. aureus to identify amino acid substitutions affecting the functional properties of different DnaK alleles
ARV-1502 binds with sequence motif YLPRP in a forward binding mode to the conventional substrate binding cleft of DnaK occupying the hydrophobic pocket (Zahn et al, 2013). These are the residues in pyrrhocoricin that cannot be substituted without a loss in antimicrobial activity against Enterobacteriaceae
Summary
Proline-rich antimicrobial peptides (PrAMP) have been intensively studied with special emphasis on their mode of action, protease stability, and toxicity (Scocchi et al, 2011; Hansen et al, 2012; Li et al, 2014; Böttger et al, 2017). ARV-1502 binds via residues YLPRP to the hydrophobic pocket of the 27 kDa C-terminal substrate binding domain (SBD) of DnaK influencing its functional activity (Zahn et al, 2013). These residues are involved in the in vitro antimicrobial activity of pyrrhocoricin analogs (Cassone et al, 2008). The nucleotide exchange factor GrpE supports this step initiating a new cycle (Harrison and Kuriyan, 1997; Brehmer et al, 2001), which starts with Hsp chaperone transferring a peptide or a protein to the opened SBD of DnaK (Han and Christen, 2003). The relationship between the influence on the protein refolding activity and the antimicrobial activity of the novel peptides was thoroughly examined
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