Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2012 Mar 16;287(12):8944-53. doi: 10.1074/jbc.M111.332205. Epub 2012 Jan 23.
Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.
人源髓系α-防御素（HNPs）在天然宿主防御中发挥多种作用。先前研究表明，HNP1 中的色氨酸 26 残基（Trp-26）对其杀伤金黄色葡萄球菌（S. aureus）、抑制炭疽致死因子（LF）、结合 HIV-1 的 gp120、二聚化以及进一步自组装的能力非常重要。为了更深入地了解二聚化的功能意义，我们将野生型 HNP1 与二聚化受损的 N-甲基化 HNP1 单体和必需的二硫键连接的 HNP1 二聚体进行了比较。这些修饰的结构影响通过 X 射线晶体学分析得到了证实。与之前研究的 W26A 突变一样，Ile-20 的 N-甲基化极大地降低了 HNP1 杀伤金黄色葡萄球菌、抑制 LF 和结合 gp120 的能力。重要的是，这种修饰对 HNP1 杀伤大肠杆菌的能力几乎没有影响。W26A 和 MeIle-20 突变协同损害防御素的活性。N 端共价连接挽救了 W26A-HNP1 抑制 LF 的能力，但未能恢复其对金黄色葡萄球菌杀伤的缺陷。表面等离子体共振研究表明，Trp-26 介导了 HNP1 单体和典型二聚体与固定化 HNP1、LF 和 gp120 的结合，并且还表明 HNP1 通过 Ile-20 和 Leu-25 可能存在四聚化的模式。这项研究表明，二聚化有助于 HNP1 的许多不同活性中的一些，但不是全部。
