Structure/function--HIV/SIV EnvelopeTransmembrane Gp41

结构/功能--HIV/SIV包膜跨膜Gp41

• 批准号: 7007430
• 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/7007430
• 关键词: Escherichia coli; HIV envelope protein gp120; HIV envelope protein gp41; X ray crystallography; chimeric proteins; conformation; glycoprotein structure; membrane fusion; membrane proteins; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein folding; protein isoforms; protein purification; protein structure function; recombinant proteins; simian immunodeficiency virus; stable isotope; structural biology; transfection

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.

背景资料：人（HIV）和相关猿（SIV）免疫缺陷病毒的包膜糖蛋白被合成为gp 160前体，其被加工成两种非共价结合的糖蛋白：gp 120和gp 41。gp 120通过结合细胞受体CD 4和趋化因子辅助受体介导病毒进入宿主细胞，两者都位于宿主细胞表面。这种结合诱导跨膜gp 41的构象变化，这有利于病毒和宿主膜之间的膜融合。在分子水平上理解这些过程可能会导致抑制HIV感染的直接手段。HIV pg 41和密切相关的SIV gp 41是高度糖基化的跨膜蛋白。位于病毒膜外表面的胞外域通过N-末端融合结构域直接介导膜融合事件。gp 41胞外域的NMR和X-射线结构都已得到解决。通过这两种方法确定的结构是棒状三聚体，其包含在中心组装为卷曲螺旋的三个平行N-末端α-螺旋，在外部包装有三个反平行C-末端α-螺旋，具有连接内螺旋和外螺旋的高度柔性环。 结果如下：为了进一步了解gp 41的功能，我们在细菌中表达了目前没有结构信息的蛋白质区域，即极疏水的N-末端融合肽、跨膜区和长胞质结构域。所产生的蛋白质正在通过新的NMR技术进行研究（A. Bax），设计用于膜蛋白和肽的结构分析。先前确定了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。我们正在制备螺旋N-末端融合结构域的位点特异性突变体，其废除融合过程以关联活性和结构。 在其他研究中，我们检查了HIV gp 41胞外域的突变体，这些突变体对靶向膜融合的新型肽抑制剂具有抗性。我们已经表明，耐药突变增加了gp 41胞外域的热力学稳定性，这些结果为耐药的潜在机制提供了新的见解。 意义和未来方向：更详细的结构测定的整个gp 41蛋白将允许一个更合理的方法来设计新的肽抑制剂。