Structure/function--HIV/SIV EnvelopeTransmembrane Gp41

背景：人类免疫缺陷病毒(HIV)和猴相关免疫缺陷病毒(SIV)的包膜糖蛋白被合成为gp160前体，它们被加工成两个非共价结合的糖蛋白：gp120和gp41。Gp120通过与位于宿主细胞表面的细胞受体CD4和趋化因子辅助受体结合来介导病毒进入宿主细胞。这种结合诱导跨膜gp41的构象变化，从而促进病毒和宿主膜之间的膜融合。在分子水平上了解这些过程可能会导致抑制艾滋病毒感染的直接方法。HIV pg41和与SIV密切相关的gp41是高度糖基化的跨膜蛋白。位于病毒膜外表面的胞外区通过N端融合结构域直接介导膜融合事件。Gp41胞外结构域的核磁共振和X-射线结构都得到了解决。两种方法确定的结构都是由三个平行的N-末端α-螺旋组成的棒状三聚体，在中心以螺旋形式组装，在外部包装三个反平行的C-末端α-螺旋，内螺旋和外螺旋之间有高度灵活的环连接。 结果：为了进一步了解gp41的功能，我们在目前尚无结构信息的细菌区域表达了gp41，即极端疏水的N末端融合肽、跨膜区和长胞浆区。生产的蛋白质正在用新的核磁共振技术(A.Bax)进行研究，该技术是为膜蛋白和多肽的结构分析而设计的。先前已经确定了细胞质结构域的膜相关区域的结构，现在已经确定了N-末端融合结构域的结构。我们正在准备螺旋N末端融合结构域的定点突变体，这些突变体取消了融合过程，以便将活性和结构联系起来。 在其他研究中，我们检查了HIV gp41胞外区的突变，这些突变对针对膜融合的新型多肽抑制剂具有抗药性。我们已经证明，抗性突变增加了gp41胞外区的热力学稳定性，这些结果为了解抗性的潜在机制提供了新的见解。 意义和未来方向：对整个gp41蛋白进行更详细的结构测定将使设计新型多肽抑制剂的方法更加合理。

Background: The envelope glycoprotein of the human (HIV) and related simian (SIV) immunodeficiency virus are synthesized as gp160 precursors which are processed into two non-covalently associated glycoproteins: gp120 and gp41. The gp120 mediates viral entry into the host cell by binding to the cellular receptor CD4 and a chemokine coreceptor, both of which are located on the host cell surface. This binding induces conformational changes in the transmembrane gp41, which facilitates membrane fusion between the viral and host membranes. An understanding of these processes at the molecular level may lead to a direct means of inhibiting HIV infection. HIV pg41, and the closely related SIV gp41, are heavily glycosylated transmembrane proteins. The ectodomain region, located on the outer surface of the viral membrane directly mediates membrane fusion events via an N-terminal fusion domain. Both the NMR and X-ray structures of the gp41 ectodomain have been solved. The structure determined by both methods is a rod-like trimer comprising three parallel N-terminal alpha-helices assembled as a coiled-coil in the center with three antiparallel C-terminal alpha-helices packed on the outside with highly flexible loops connecting the inner and outer helices. Results: To gain further insight into gp41 function we have expressed in bacteria regions of the protein for which there is currently no structural information, namely, the extremely hydrophobic N-terminal fusion peptide, the transmembrane region and the long cytoplasmic domain. The proteins produced are being studied by new NMR techniques (A. Bax) designed for the structural analysis of membrane proteins and peptides. The structure of a membrane associated region of the cytoplasmic domain was previously determined and the structure of the N-terminal fusion domain has now been determined. We are preparing site-specific mutants of the helical N-terminal fusion domain which abrogate the fusion process in order to correlate activity and structure. In other studies we have examined mutants of the HIV gp41 ectodomain which are resistant to the new class of peptide inhibitors targeted against membrane fusion. We have shown that the resistance mutations increase the thermodynamic stability of the gp41 ectodomain and these results provides new insight into the potential mechanism of resistance. Significance and future direction: More detailed structure determinations of the whole gp41 protein will allow a more rational approach to the design of novel peptide inhibitors.