title: Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus.
abstract: Human α-defensin 5 (HD5, HD5(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(3)-Cys(31), Cys(5)-Cys(20), Cys(10)-Cys(30)) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser(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(ox) against this Gram-positive bacterial strain. This observation supports the notion that the HD5(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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Yoshitha A
authors.initials: YA
authors.lastname: Wanniarachchi
authors.forename: Piotr
authors.initials: P
authors.lastname: Kaczmarek
authors.forename: Andrea
authors.initials: A
authors.lastname: Wan
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Nolan
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism*; D023083:beta-Defensins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; C465593:DEFB105A protein, human; D004220:Disulfides; D023083:beta-Defensins