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（HIV）和相关的Simian（SIV）免疫缺陷病毒的包膜糖蛋白合成为GP160前体，这些前体被处理成两个非共价相关的糖蛋白：GP120和GP41。 GP120通过结合细胞受体CD4和趋化因子共肽剂，介导病毒进入宿主细胞，它们都位于宿主细胞表面。这种结合诱导跨膜GP41的构象变化，这促进了病毒和宿主膜之间的膜融合。在分子水平上对这些过程的理解可能导致抑制HIV感染的直接手段。如HIV PG41和密切相关的SIV GP41，是大量糖基化的跨膜蛋白对其高分辨率结构的测定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最重要的区域是位于病毒膜外表面的胞外域区域，该区域直接介导膜融合事件。 HIV和SIV GP41胞外域都在大肠杆菌中表达。对于SIV GP41，NMR和X射线结构均已解决。结果：已经确定了SIV GP41胞外域的高分辨率X射线（Hyde/Wang）和NMR（Clore/Gronenborn/Caffrey）结构。通过两种方法确定的结构是一种类似杆状的三聚体，其中包括三个平行的N端A螺旋，在中心盘绕的螺旋线组装，带有三个反平行的C端A螺旋，在外部堆积了三个反平行的C端A螺旋，并具有高度柔韧的环，连接了内部和外部螺旋。连接内部和外螺旋的环区域虽然在X射线模型中并未明确解析，但已在NMR结构中建模，允许建立内螺旋和外部螺旋之间的连接。该区域似乎是导致三聚体在生理pH值下的低溶解度，从而导致骨料形成。我们最近的工作使我们假设GP41的这些高分子聚集体负责HIV相关的神经系统损害和痴呆。 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，尽管它与HIV基质蛋白（核素的成分）以及各种宿主细胞因子相互作用。意义和未来方向：GP41外生域的高分辨率结构确定将允许对新型肽抑制剂的设计进行更合理的方法。基于GP41的物理特性，已经提出了涉及部分相关的蛋白质复合物的膜融合模型。这些模型还可以为靶向GP41提供有用的线索。膜融合的更完整的图像将需要在GP41上进行更广泛的结构工作，尤其是那些参与与GP120相互作用的区域。已经表达并正在测试中表达了各种工程GP120蛋白的表达，包括被认为与GP41相互作用的N和C末端结构域的表达。Summary：人类免疫缺陷病毒（HIV）表面上的包膜蛋白（HIV），该病毒介导该病毒进入其宿主细胞的介导了细菌中的培养基细胞中已经表达了细菌的重点。纯化包膜蛋白并研究了结构。希望对这些蛋白质的分子细节的理解，对病毒生命周期至关重要，将有助于开发试剂和方法来阻止其功能。 - HIV/SIV，包膜糖蛋白GP41，膜融合，重组蛋白表达