Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans
Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans
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DOI：
10.1021/acs.biochem.7b00348
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发表时间：
2017-08-15
期刊：
影响因子：
2.9
通讯作者：
Franz, Katherine J.
中科院分区：
文献类型：
--
作者：
Conklin, Steven E.;Bridgman, Emma C.;Franz, Katherine J.
已知组胺素家族的富含组氨酸的唾液肽在体外结合铜（Cu）和其他金属离子;然而，这些相互作用的细节知之甚少，并且它们对体内抗真菌活性的影响尚未建立。在这里，我们表明，铜的可用性暴露于白念珠菌组胺素-5（Hist-5）调节其抗真菌活性。抗真菌药敏试验表明，共同治疗组蛋白-5与铜改善EC 50从类似5到类似1 μ M，而共同治疗与高亲和力铜特异性螯合剂废除抗真菌活性。分光光度滴定显示两个先前未识别的Cu（I）结合位点，其在pH 7.4下的表观Kd值与20 nM相似，并证实了在Hist-5 N-末端的高亲和力Cu（II）结合位点，其表观Kd与8 pM相似。对一系列His-至-Ala全长和截短的Hist-5肽的评价鉴定了相邻的His残基（bis-His）作为Cu（I）结合的关键锚，其中第三配体的存在通过X射线吸收光谱揭示。截短肽本身不能有效抑制C.白色念珠菌，但用补充Cu处理导致EC 50值下降至接近5 μ M，接近全长Hist-5的EC 50值。肽的功效取决于完整的双-His位点，并与Cu（I）亲和力相关。总之，这些结果建立了新的结构-功能关系，连接特定的组氨酸残基与铜结合亲和力和抗真菌活性，并提供了进一步的证据，参与金属在调节这些抗真菌肽的生物活性。
The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro; however, the details of these interactions are poorly understood, and their implications for in vivo antifungal activity have not been established. Here, we show that the 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 similar to 5 to similar to 1 mu M, 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 K-d values at pH 7.4, similar to 20 nM, and confirmed a high-affinity Cu(II)-binding site at the Hist-5 N-terminus with an apparent K-d of similar to 8 pM. Evaluation of a series of His-to-Ala full-length and truncated Hist-5 peptides identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, with the presence of a third ligand revealed by X-ray absorption spectroscopy. On their own, the truncated peptides were ineffective at inhibiting the growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to similar to 5 mu M, 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 of the involvement of metals in modulating the biological activity of these antifungal peptides.