Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5[subscript ox] to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys[superscript 3]-Cys[superscript 31], Cys[superscript 5]-Cys[superscript 20], Cys[superscript 10]-Cys[superscript 30]) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser[superscript hexa]], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S–S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5[subscript ox] against this Gram-positive bacterial strain. This observation supports the notion that the HD5[subscript ox] mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem.284, 29180−29192] and that the native disulfide array is a requirement for its activity against S. aureus.Massachusetts Institute of Technology. Biophysical Instrumentation Facility ((NSF- 0070319)Massachusetts Institute of Technology. Biophysical Instrumentation Facility (NIH GM68762