The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance

Abstract

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg7 or Glu15 residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirmation that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg7 and Glu15 substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.

Invariant Gly Residue Is Important for α-Defensin Folding, Dimerization, and Function

“…There are a total of seven invariant residues in all known mammalian ␣-defensins: six cysteines that form the three intramolecular disulfides and Gly 17 (HNP1 numbering) that constitutes part of the ␤-bulge structure. In addition, a salt bridge between Arg 5 and Glu 13 (HNP1 numbering) exists in most mammalian ␣-defensins, contributing a great deal to ␣-defensin folding and in vivo stability (45)(46)(47)(48), but little to ␣-defensin functionality (10). Although disulfide bonding is critical for ␣-defensin biosynthesis (49) and the vast majority of reported ␣-defensin functions (10), the structural and functional basis for the strict conservation of Gly 17 largely remains elusive.…”

Rattusin, an Intestinal α-Defensin-Related Peptide in Rats with a Unique Cysteine Spacing Pattern and Salt-Insensitive Antibacterial Activities

“…The macrocyclic -defensins are exceptions in that their bactericidal activities are nearly abolished when they are reduced (23). Although the integrity of disulfide bonds and spatial structure does not determine the antibacterial activities of most defensins per se, the disulfide arrays are required for other biological functions, such as chemotactic activities (21) and resistance to proteolysis (47). However, it remains unknown whether reduction of the disulfide bonds of rattusin impact its nonbactericidal activities.…”

Functional Determinants of Human Enteric α-Defensin HD5

“…Many of the prior studies have been focused on two model systems: HNP1 and ␣-defensins from mouse crypts, also known as cryptdins (50). Collectively, these studies have provided important insights into the structural and functional roles of disulfide bonding (51,52), cationicity (53,54), and conserved elements, such as the Arg-Glu salt bridge (55)(56)(57)(58) and invariant Gly residue (59,60) in the action of ␣-defensins. Recently, a comprehensive Ala scanning mutagenesis of HNP1 has discovered that a hydrophobic residue near the C terminus, Trp 26 , governs the ability of this ␣-defensin to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind HIV-1 envelope glycoprotein gp120 (61); methylation of a peptide bond at the putative dimer interface of HNP1 debilitates its dimerization and is functionally detrimental (62).…”