Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120
Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120
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DOI：
10.1074/jbc.m702025200
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发表时间：
2007-09-21
影响因子：
4.8
通讯作者：
Subramaniam, Sriram
中科院分区：
文献类型：
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作者：
Bennett, Adam;Liu, Jun;Subramaniam, Sriram
确定人类免疫缺陷病毒（HIV）中和的结构决定因素是合理药物和疫苗设计的重要组成部分。我们使用冷冻电子断层扫描和原子力显微镜来表征一种非常有效的HIV中和蛋白D1 D2-IG alpha tp（缩写为D1 D2-IgP）的结构，D1 D2-IgP是一种多价抗体构建体，其呈现十二聚体CD 4代替Fab区域。我们发现，D1 D2-IgP具有一种新的结构，其抗体臂比密切相关的IgM具有更大的灵活性。使用猿免疫缺陷病毒与D1 D2-IgP的复合物，我们提出了明确的证据，D1 D2-IgP可以交联相同病毒和相邻病毒的表面刺突。观察到的与病毒包膜刺突的结合是特异性CD 4-gp 120相互作用的结果，因为用MICA-IgP没有观察到结合，MICA-IgP是一种与DlD 2-IgP相同的构建体，除了主要组织相容性复合体I类相关链A（云母）替代了CD 4部分。CD 4介导的结合也与破裂病毒的比例显著升高相关。灭活的与CD 4配体的gp 120-gp 41刺突的比率可以远大于1：1，因为在交联病毒的紧密并置表面上的所有gp 120-gp 41刺突由于病毒间刺突交联而不能接近靶细胞表面并介导进入。这些结果暗示柔性而不是空间体积或多价本身作为DlD 2-IgP的极端效力的结构解释，因此表明在柔性支架上呈现的多价作为小分子HIV进入抑制剂的关键设计标准。
Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig alpha tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that DlD2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to DlD2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of DlD2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors.