Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus

Biochemistry. 2011 Sep 20;50(37):8005-17. doi: 10.1021/bi201043j. Epub 2011 Aug 23.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Disulfides / chemistry*
  • Disulfides / metabolism*
  • Gene Deletion*
  • Humans
  • Molecular Sequence Data
  • Mutation / genetics*
  • Protein Structure, Secondary
  • Staphylococcus aureus / drug effects
  • Staphylococcus aureus / metabolism*
  • beta-Defensins / chemistry*
  • beta-Defensins / deficiency
  • beta-Defensins / genetics*

Substances

  • Anti-Bacterial Agents
  • DEFB105A protein, human
  • Disulfides
  • beta-Defensins