Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4

Abstract

The ␣-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines ␣-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell ␣-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the ␣-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining ␣-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Trp-26 Imparts Functional Versatility to Human α-Defensin HNP1

“…Some Structurally Conserved Elements, although Essential for Defensin Biosynthesis, Do Not Contribute to HNP1 FunctionEarlier structure-activity studies of ␣-defensins, including our own, typically focused on their conserved structural elements (23)(24)(25)(26)(27)(28), including an invariant Gly-17, a salt bridge between Arg-5 and Glu-13 (using HNP1 numbering), and disulfide bonding. Gly-17, part of an atypical ␤-bulge structure, is critical for ␣-defensin folding, whereas the salt bridge stabilizes ␣-defensins to prevent their in vivo degradation by proteases.…”

“…By contrast, the loss of disulfide bonding causes collapse of defensin tertiary structure and indiscriminately inactivates ␣-defensins in several biological assays such as S. aureus killing, LF inhibition, and gp120 binding (35), except for killing certain Gram-negative bacteria such as Escherichia coli. Disulfide bonding is important for defensin biosynthesis and in vivo stability (23). Despite its vital role in maintaining defensin structure, disulfide bonding itself is rarely targeted for mutational analysis as it generally confers little functional specificity.…”

“…Prior mutational studies of ␣-defensins focused primarily on conserved elements such as disulfide bonding (23,24), an invariant Gly residue (25), a conserved salt bridge (26 -28), and the often abundant but less conserved Arg residues (29,30). However, the loss of many of those conserved structural elements in ␣-defensins is often functionally inconsequential in vitro.…”

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).…”

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

“…Moreover, until now, cysteine-deleted mutants [10,11], salt bridge-deficient mutants [9,12], positive-to-negative charge-reversal mutants [13] and an Arg-to-Lys mutant of Crp4 [14,15] have been studied to elucidate the role of the conserved biochemical features of α-defensins. In previous studies, the wild-type and mutants of Crp4 were prepared using the Escherichia coli expression system [8][9][10][11][12][13][14][15]. In this expression system, the recombinant wild-type and Crp4 mutants are expressed in E. coli as N-terminally linked, 6-histidine-tagged fusion peptides.…”