Structure/function Of Hiv/siv Envelope Transmembrane Gly

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。由于HIVpg41和SIVgp41是高度糖基化的跨膜蛋白，因此确定它们的高分辨结构是一个非常困难的问题。在增量的方法中，我们正在研究gp41的(非糖基化)功能结构域的结构。Gp41最重要的区域是位于病毒膜外表面的胞外区，它直接介导膜融合事件。HIV和SIV gp41胞外区均已在大肠杆菌中表达。对于SIV gp41，已经解决了核磁共振和X射线结构。结果确定了SIV gp41胞外区(e-gp41)的高分辨X射线(Hyde/Wang)和核磁共振(Clore/GronenBorn/Caffrey)结构。两种方法确定的结构都是棒状三聚体，由三个平行的N-末端a-螺旋在中心组装成螺旋线圈，三个反平行的C-末端a-螺旋排列在外部，内外螺旋之间有高度灵活的环连接。连接内螺旋和外螺旋的环区，虽然在X射线模型中没有明确解析，但已经在核磁共振结构中建模，允许建立内螺旋和外螺旋之间的连接。这一区域似乎与三聚体在生理pH值下的低溶解度有关，从而导致聚集体的形成。E-GP4的聚集体由7-70个三聚体组成，它们通过涉及环区的聚集体内部的相互作用而结合在一起。在确诊为痴呆症的HIV-1感染患者的大脑中，HIV-1 gp41以细胞外聚集体的形式积累。据推测，e-gp41的高分子聚集体与HIV相关的神经损伤和痴呆有关。来自e-gp41 C-末端螺旋的多肽在NM浓度下抑制融合。有人提出，这些多肽在融合状态下(由gp120-CD4相互作用产生)与暴露的N-末端螺旋结合。我们已经解剖了e-gp41分子，形成了N末端和C末端的多肽结构域。对这些多肽的结构研究和生化测量将有助于更清楚地了解它们的抑制活性。Gp41蛋白的其他功能区域在细菌中也有表达，包括细胞质区域，该区域虽然与HIV基质蛋白(核衣壳的一个组成部分)以及各种宿主细胞因子相互作用。正在对这些化合物进行表征，以便在核磁共振研究中使用。意义和未来方向gp41胞外结构域的高分辨率结构测定将为设计新型多肽抑制剂提供更合理的途径。基于gp41的物理性质，提出了涉及部分结合蛋白复合体的膜融合模型。这些模型也可能为靶向gp41提供有用的线索。要想更完整地了解膜融合，将需要在gp41上进行更广泛的结构工作，特别是那些参与与gp120相互作用的区域。各种工程化gp120蛋白的表达，包括被认为与gp41相互作用的N-末端和C-末端结构域，已经被表达并正在进行测试。

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. As HIV pg41, and the closely related SIV gp41, are heavily glycosylated transmembrane proteins determination of their highresolution structures is a very difficult problem. In an incremental approach, we are studying the structure of (non-glycosylated) functional domains of gp41. The most important region of gp41 is the ectodomain region, located on the outer surface of the viral membrane, which directly mediates membrane fusion events. Both HIV and SIV gp41 ectodomains have been expressed in E.coli. For the SIV gp41, both the NMR and X-ray structures have been solved. Results High-resolution X-ray (Hyde/Wang) and NMR (Clore/Gronenborn/Caffrey) structures of the SIV gp41 ectodomain (e-gp41) have been determined. The structure determined by both methods is a rod-like trimer comprising three parallel N-terminal a-helices assembled as a coiled-coil in the center with three antiparallel C-terminal a-helices packed on the outside with highly flexible loops connecting the inner and outer helices. The loop region connecting the inner and outer helices, although not clearly resolved in the X-ray model, has been modeled in the NMR structure allowing the connectivity's between inner and outer helices to be established. This region appears responsible for the low solubility of the trimer at physiological pH values resulting in aggregate formation. Aggregates of e-gp4 are composed of between 7-70 trimers, which are associated by interactions at the interior of the aggregate involving the loop region. In the brains of HIV-1 infected patients diagnosed with dementia, HIV-1 gp41 accumulates as an extracellular aggregate. It is postulated that the high molecular aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia. Peptides from the C-terminal helix of e-gp41 inhibit fusion at nM concentrations. It has been proposed that the peptides bind to the exposed N-terminal helix in the fusogenic state (generated by the gp120-CD4 interaction). We have dissected the e-gp41 molecule making the N-terminal and C-terminal peptide domains. Structural studies and biochemical measurements of these peptides will help gain a clearer picture of their inhibitory activity. Other functional regions of the gp41 protein have been expressed in bacteria, including the cytoplasmic domain, which is though to interact with the HIV matrix protein (a component of the nucleocapsid) as well as various host cellular factors. These are being characterized for use in NMR studies. Significance and future direction The high-resolution structure determinations of the gp41 ectodomain will allow a more rational approach to the design of novel peptide inhibitors. Based on the physical properties of the gp41, models of membrane fusion have been proposed which involve partially associated protein complexes. These models may also provide useful clues for targeting gp41. A more complete picture of membrane fusion will require more extensive structural work on gp41, especially those regions involved in mediating interaction with gp120. The expression of various engineered gp120 proteins, including the N- and C-terminal domains which are thought to interact with gp41, have been expressed and are being tested.