Key Laboratory of Fermentation Engineering, Ministry of Education, College of Bioengineering, Hubei University of Technology, Wuhan, China; Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA.
Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA.
Biochim Biophys Acta Biomembr. 2019 Apr 1;1861(4):835-844. doi: 10.1016/j.bbamem.2019.01.007. Epub 2019 Jan 15.
Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.
防御素是先天免疫中一类具有免疫调节特性的阳离子抗菌肽。典型的人α-防御素，也称为人中性粒细胞肽 1-3 或 HNP1-3，因其结构、功能和作用机制而得到广泛研究，但对 HNP4 的了解甚少——HNP1-3 的“远房表亲”，其丰度要低得多。在这里，我们报告了对 HNP4 的系统突变分析，涉及它对大肠杆菌和金黄色葡萄球菌的抗菌活性、对炭疽致死因子 (LF) 的抑制活性以及对 LF 和 HIV-1 gp120 的结合活性。除了 9 个保守且结构重要的残基 (6xCys、1xArg、1xGlu 和 1xGly) 外，HNP4 的其余 24 个残基分别突变为 Ala。G23A-HNP4 和 T27A-HNP4 的晶体结构被确定，均表现出与野生型 HNP4 相同的二硫键稳定的典型α-防御素二聚体。与 HNP1-3 不同，HNP4 优先杀死革兰氏阴性菌，这一特性主要归因于三个聚集的阳离子残基 Arg10、Arg11 和 Arg15。阳离子簇对于 HNP4 杀死金黄色葡萄球菌、抑制 LF 和与 LF 和 gp120 结合也很重要。然而，F26A 虽然对大肠杆菌的杀伤作用无足轻重，但比任何其他突变都更具破坏性。同样，将 Leu20 甲基化以破坏 HNP4 二聚体对大肠杆菌杀伤几乎没有影响，但显著降低了 HNP4 杀死金黄色葡萄球菌、抑制 LF 和与 LF 和 gp120 结合的能力。我们的发现揭示了 HNP4 功能的分子决定因素，完成了所有人中性粒细胞α-防御素的结构和功能关系图谱。
