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

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... tericidal 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 pro-Crp4 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.