Structure-Activity Relationships in Defensin Dimers

Abstract

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a ␤-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mM NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have ␤-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter-and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.␤-Defensins are members of the antimicrobial peptide family that are important components of the mammalian innate immune response (1, 2). In addition to potent bactericidal activity, they can also act upon T lymphocytes and immature dendritic cells thus playing key roles in adaptive immunity (3). They are produced as prepropeptides and are processed to a mature secreted peptide, which have six canonical cysteine residues with spacing and intramolecular disulfide bridge connectivity (Cys ) distinct from the similar ␣-defensins (4). A recent bioinformatic analysis of the human and mouse genomes revealed a significantly higher number of ␤-defensin genes than previously thought, although the function of each individual defensin has still to be determined in vivo (5).Two strong lines of evidence have demonstrated the significance of the...

Studies of the Biological Properties of Human β-Defensin 1

“…Thus, the structural framework of ␤-defensin molecules appears to tolerate a substantial variability of amino acid sequences and is also robust to the environmental changes (for instance, pH of crystallization conditions varies between 4.6 and 8.5). This observation is consistent with the existence of many proteins adopting a defensin-like fold despite rather low amino acid sequence homology (52). Consistent with previous results of studies in solution (44,45), formation of distinct oligomers of hBD1 was not observed for any of the 10 structures.…”

Why Is the Arg5-Glu13 Salt Bridge Conserved in Mammalian α-Defensins?

“…It is plausible that the conserved salt bridge affords these ␣-defensins proteolytic resistance to their processing enzymes. In fact, Ouellette and colleagues (56) demonstrated that structural stability afforded by disulfide bonding in mouse ␣-defensins (cryptdins), whereas not required for their antimicrobial function as shown in several ␤-defensins (28,58,59), is essential for preventing in vivo proteolytic degradation by their processing enzyme, the metalloproteinase matrilysin (38,60). Our work has established the importance of two highly conserved, oppositely charged residues in maintaining ␣-defensin in vivo stability and thus biological functions of these multifaceted immunological molecules.…”

Analysis and Separation of Residues Important for the Chemoattractant and Antimicrobial Activities of β-Defensin 3

“…Defb14, Defb14-1c v , and HBD3 were obtained from Chemical Synthesis Services-Albachem Ltd. (Gladsmuir, UK). Disulfide connectivities were determined by proteolysis and peptide mass mapping as previously published (10,20). Briefly, a 50 M solution of the peptide was subjected to proteolytic cleavage with a combination of trypsin and chymotrypsin (Sigma) for HBD3.…”

“…Antimicrobial Assays-These assays were carried out as previously described (20). Briefly, test organisms were grown to midlogarithmic phase in Iso-Sensitest broth (Oxoid) growth medium and then diluted to 1-5 ϫ 10 6 colony-forming units/ml in 10 mM potassium phosphate containing 1% (v/v) Iso-Sensitest broth, pH 7.4.…”

“…The gene for this five-cysteine peptide (Defr1) is a C57Bl/6 polymorphism of the six-cysteine peptide encoding Defb8 in other inbred strains of mice. The Defr1 peptide forms a covalent, disulfide-linked dimer with potent antimicrobial activity, which is diminished upon reduction (20,21). This unique fivecysteine defensin is also active as a chemoattractant.…”