Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation
Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation
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DOI：
10.1089/aid.2012.0286
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发表时间：
2013-04-01
影响因子：
1.5
通讯作者：
Burakoff, Steven J.
中科院分区：
文献类型：
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作者：
Jin, Yong-Jiu;Cai, Catherine Yi;Burakoff, Steven J.
HIV-1 Nef 是 HIV-1 毒力和艾滋病快速发展所必需的辅助蛋白。 Nef 通过将 CD4 和其他几种细胞表面受体连接到网格蛋白接头蛋白 AP2 来促进病毒复制和感染，从而导致与 Nef 相互作用的受体的内化和降解。我们研究了 Nef 如何通过其 C 末端柔性环（C 环）中的双亮氨酸基序与 AP2 相互作用来介导组成型受体内吞作用，而跨膜受体的 AP2 结合通常会导致平衡（循环）内吞作用。我们的结果表明，除了二亮氨酸基序之外，Nef C 环中还有第二个基序参与 Nef-AP2 相互作用。当 Nef C 环 (LL(165)HPMSLHGM(173)) 疏水区域的残基突变为碱性残基 K/R 或酸性残基 E/D 或刚性残基 P，或者当 M168L170、L170H171 或 G(172)M(173) 突变为 AA 时，Nef 介导的 CD4 下调会受到损害。 Pull-down 测定表明 AP2 不与不下调 CD4 的 Nef 突变体共沉淀。 Nef C 末端柔性环与 AP2 复合物的分子模型表明，M168L170 占据 AP2 sigma 2 亚基中的一个口袋。我们的数据提出了 Nef-AP2 相互作用的新模型，其中 Nef C 环中的疏水区域具有双亮氨酸 (L164L165) 基序和 M168L170 基序与 AP2(sigma 2) 结合，而酸性基序 E-174 和 D-175 与 AP2(α) 结合，这解释了 Nef 如何通过柔性环将 CD4 连接到 AP2 以实现组成型 CD4 下调。
HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL(165)HPMSLHGM(173)) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G(172)M(173) was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 sigma 2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(sigma 2), while the acidic motif E-174 and D-175 binds to AP2(alpha), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.