Structural Insights into Bactericidal Activity of Corneal Epithelial Keratin-Derived Antimicrobial Peptides (KDAMPs)

Abstract

 

Purpose

 

KDAMPs are a family of glycine-rich C-terminal fragments derived constitutively from keratin 6A in human corneal epithelial cells. Synthetic analogs of KDAMPs (10-36 amino acids) kill various bacteria of both Gram types including Pseudomonas aeruginosa, a major corneal pathogen. Interestingly, KDAMPs that differ by just a few residues in their primary sequences showed distinctive antimicrobial activity spectra. Here, we determined the 3D NMR solution structures of 10-mer and 19-mer in SDS micelles (a widely accepted model mimicking bacterial membrane) to gain insights into their mode of action.

 

Methods

 

NMR spectra of 10-mer and 19-mer were acquired with Bruker Biospin Avance 900 MHz spectrometer and Varian Inova 600MHz spectrometer respectively. Data were processed with NMRPipe. 2D ¹H-¹H DQF-COSY, ¹H-¹H TOCSY and ¹H-¹H NOESY spectra were obtained in an aqueous solution containing 10% D₂O, 3.4 mM peptide and deuterated SDS in a peptide-to-SDS molar ratio of 1:60 at pH 4.5 and 298K. Tertiary structures in SDS micelles were determined by H^α secondary shift analysis, NOE analysis, and chemical shift-based structural calculation using the CS23D program.

 

Results

 

While glycine-rich peptides do not commonly form secondary structures, the 10-mer and 19-mer of KDAMPs revealed some helical conformations. Specifically, Gly2-Gly7 of 10-mer, as well as Gly5-Val10 and Ser14-Lys18 of 19-mer compose α-helices. Overall, a helix-loop-helix topology was observed in 19-mer, while the only helix in 10-mer aligns with the N-terminal helix in 19-mer. The major differences between the two structures were found to be the additional C-terminal helix and the flexible loop composed of three glycine residues (Gly11-Gly13) between the two helices in 19-mer.

 

Conclusions

 

The similarity in the 10-mer and 19-mer structures suggests their similar bactericidal mechanisms. Yet previous findings indicate that there are additional bacterial species to which 19-mer is more potent as compared to 10-mer (e.g. Streptococcus pyogenes). Therefore it is possible that KDAMPs exert killing effect on some bacteria with only the N-terminal helix, while both helices and/or the loop are required to kill other bacteria. The structural domains probably play a role in defining the activity spectra of specific KDAMPs.

 

 

Structural alignment of 10-mer (yellow) and 19-mer (cyan).

 

Keywords: 482 cornea: epithelium • 664 pseudomonas • 422 antibiotics/antifungals/antiparasitics