Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro, but the details of these interactions are poorly understood and their implications on in vivo antifungal activity have not been established. Here, we show that availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC50 from ~5 µM to ~1 µM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrations revealed two previously unrecognized Cu(I) binding sites with apparent Kd values at pH 7.4 ~ 20 nM, and confirmed a high-affinity Cu(II) binding site at the Hist-5 N-terminus with apparent Kd ~ 8 pM. Evaluation of a series of His-to-Ala peptides containing the first 12 residues of Hist-5 identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, and the presence of a third ligand was revealed by X-ray absorption spectroscopy (XAS). On their own, the truncated peptides were ineffective at inhibiting growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ~ 5 µM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu-binding affinity and antifungal activity, and provide further evidence for the involvement of metals in modulating the biological activity of these antifungal peptides.