Functional Determinants of Human Enteric α-Defensin HD5 CRUCIAL ROLE FOR HYDROPHOBICITY AT DIMER INTERFACE
Functional Determinants of Human Enteric α-Defensin HD5 CRUCIAL ROLE FOR HYDROPHOBICITY AT DIMER INTERFACE
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DOI：
10.1074/jbc.m112.367995
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发表时间：
2012-06-22
影响因子：
4.8
通讯作者：
Lu, Wuyuan
中科院分区：
文献类型：
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作者：
Rajabi, Mohsen;Ericksen, Bryan;Lu, Wuyuan
人类α -防御素是自结合成二聚体和高阶低聚物的阳离子肽。它们结合蛋白质毒素，如炭疽致死因子（LF），并杀死细菌，包括大肠杆菌和金黄色葡萄球菌，以及其他功能。人类α -防御素家族有6个成员：4个人类中性粒细胞肽，包括HNP1； 2个肠道人类防御素，包括HD5。我们对HD5进行全面的丙氨酸扫描诱变。然后，我们用表面等离子体共振法检测LF结合，用酶动力学抑制法检测LF活性，用虚拟菌落计数法检测LF抗菌活性。大多数突变可以耐受，导致与野生型HD5相当的活性。然而，L29A突变减少了LF结合和对大肠杆菌和金黄色葡萄球菌的杀菌活性。在第29位的一系列非天然脂肪族和芳香族取代，包括氨基丁酸（Abu）和去甲亮氨酸（Nle），其疏水性与HD5的功能相关。L29Abu-HD5的晶体结构描述了二聚体界面疏水接触的减少，而Nle-29-HD5的晶体结构描述了一种平行β链的新型二聚化模式。突变Leu（29）的效果类似于HNP1的c端疏水残基Trp（26）。此外，为了进一步阐明二聚化在HD5功能中的作用，我们通过Glu（21）残基的n -甲基化生成了一个专一性单体，降低了LF结合和对金黄色葡萄球菌的抗菌活性。这些结果进一步表征了α -防御素的二聚体界面，揭示了疏水性介导的二聚化的关键作用。
Human alpha-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins, such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human alpha-defensin family: four human neutrophil peptides, including HNP1, and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However, the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine (Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel beta strands. The effect of mutating Leu(29) is similar to that of a C-terminal hydrophobic residue of HNP1, Trp(26). In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by N-methylation of the Glu(21) residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the alpha-defensins, revealing a crucial role of hydrophobicity-mediated dimerization.