STRUCTURE/FUNCTION OF HIV/SIV ENVELOPE TRANSMEMBRANE GLYCOPROTEIN GP41

HIV/SIV 包膜跨膜糖蛋白 GP41 的结构/功能

• 批准号: 6289042
• 负责人: PAUL T WINGFIELD
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289042
• 关键词: Escherichia coli; HIV envelope protein gp120; HIV envelope protein gp41; X ray crystallography; conformation; crystallization; glycoprotein structure; mutant; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein folding; protein isoforms; protein purification; protein structure function; simian immunodeficiency virus; stable isotope

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 high resolution 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 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. Our very recent work has led us to postulate these high molecular aggregates of the gp41 are responsible for HIV- associated neurological damage and dementia. 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. 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.Summary:Envelope proteins on the surface of the human immunodeficiency virus (HIV) that mediate entry of the virus into its host cell have been expressed in bacteria using recombinant DNA methods. The envelope proteins were purified and their structures studied. It is hoped that an understanding of the molecular details of these proteins, vital for the viral life cycle, will aid in the development of reagents and methods for blocking their function. - HIV/SIV, envelope glycoprotein gp41, membrane fusion, recombinant protein expression

背景：人类（HIV）和相关猿猴（SIV）免疫缺陷病毒的包膜糖蛋白被合成为gp160前体，其被加工成两种非共价相关的糖蛋白：gp120和gp41。 gp120 通过与细胞受体 CD4 和趋化因子辅助受体（两者均位于宿主细胞表面）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产生抑制艾滋病毒感染的直接方法。由于HIV pg41和密切相关的SIV gp41是高度糖基化的跨膜蛋白，确定其高分辨率结构是一个非常困难的问题。在增量方法中，我们正在研究 gp41 的（非糖基化）功能域的结构。 gp41最重要的区域是胞外域区域，位于病毒膜的外表面，直接介导膜融合事件。 HIV 和 SIV gp41 胞外域均已在大肠杆菌中表达。对于SIV gp41，NMR 和 X 射线结构均已解析。结果：SIV gp41 胞外域的高分辨率 X 射线 (Hyde/Wang) 和 NMR (Clore/Gronenborn/Caffrey) 结构已确定。两种方法确定的结构是一个杆状三聚体，包含三个平行的 N 端 a 螺旋，在中心组装成盘绕线圈，三个反平行的 C 端 a 螺旋包装在外部，并通过高度灵活的环连接内部和外螺旋。连接内螺旋和外螺旋的环区域虽然在 X 射线模型中没有明确解析，但已在 NMR 结构中进行了建模，允许建立内螺旋和外螺旋之间的连接。该区域似乎是造成三聚体在生理 pH 值下溶解度低、导致聚集体形成的原因。我们最近的工作使我们推测 gp41 的这些高分子聚集体是导致 HIV 相关神经损伤和痴呆的原因。 gp41 蛋白的其他功能区域已在细菌中表达，包括细胞质结构域，该结构域被认为与 HIV 基质蛋白（核衣壳的组成部分）以及各种宿主细胞因子相互作用。意义和未来方向：gp41胞外域的高分辨率结构测定将为新型肽抑制剂的设计提供更合理的方法。基于 gp41 的物理特性，已经提出了涉及部分相关蛋白复合物的膜融合模型。这些模型也可能为靶向 gp41 提供有用的线索。更完整的膜融合图需要对 gp41 进行更广泛的结构研究，特别是那些涉及介导与 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 端和 C 端结构域，已经表达并正在测试。 摘要：介导人类免疫缺陷病毒 (HIV) 表面的包膜蛋白病毒进入宿主细胞的过程已通过重组 DNA 方法在细菌中表达。纯化包膜蛋白并研究其结构。人们希望了解这些对于病毒生命周期至关重要的蛋白质的分子细节，将有助于开发阻断其功能的试剂和方法。 - HIV/SIV、包膜糖蛋白gp41、膜融合、重组蛋白表达