Structural study of the HIV1 gp41 coat protein

HIV1 gp41外壳蛋白的结构研究

• 批准号: 10253744
• 负责人: Adriaan Bax
• 金额: $ 99.52万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/10253744
• 关键词: 2019-nCoV; Affinity; Antibodies; Binding; C-terminal; Capsid Proteins; Cell membrane; Cells; Chemicals; Circular Dichroism; Detergents; Dissociation; Ebola; Epitopes; Equilibrium; Free Energy; Glycoproteins; HIV Envelope Protein gp120; HIV-1; Hemagglutinin; Hydrogen; Hydrophobicity; Influenza; Ionic Strengths; Lecithin; Lipid Bilayers; Lipids; Mediating; Membrane; Membrane Fusion; Micelles; Modeling; Molecular Conformation; N-terminal; Phosphorylcholine; Play; Process; Property; Proteins; Publications; Reporting; Role; Structure; Surface; Tertiary Protein Structure; Thermodynamics; Transmembrane Domain; Viral; Viral Fusion Proteins; Virus; Work; aqueous; biophysical techniques; design and construction; driving force; experimental study; gp160; influenzavirus; intermolecular interaction; melting; monomer; neutralizing antibody; prevent

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine and bicelles consisting of DMPC and dihexanoyl phosphatidylcholine suggested the importance of trimerization of gp41s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion. The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We have found that in aqueous solution the MPER fragment (gp160 residues 660-674) exists in a monomer-trimer equilibrium with an association constant in the micro-molar range. Thermodynamic analysis revealed that the association is exothermic, more favorable in D2O than H2O, and increased with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13C chemical shifts, NOE, and hydrogen exchange rates revealed that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an alpha-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state. Preliminary experiments applied to the ecto-domain of the Spike protein of SARS-CoV-2 indicate this protein contains a three-helical N-terminal heptad repeat (NHR) that is considerably more stable than the corresponding region of gp41, and the propensity for the C-terminal heptad (CHR) repeat albeit lower than that of NHR, is also substantially higher than for the CHR of gp41, whereas the propensity to trimerize for its membrane proximal region is substantially lower. Work has been submitted for publication but remains under review.

包膜糖蛋白 gp41 介导膜融合过程，使 HIV-1 病毒能够进入宿主细胞。胞外域的强脂质亲和力表明，其七肽重复区域通过直接结合脂质双层，从而降低膜融合的自由能屏障，从而在膜不稳定中发挥积极作用。在这样的模型中，gp120脱落后，N-七肽和C-七肽螺旋立即解离并分别融化到宿主细胞和病毒膜中，将不稳定的膜拉至并置，准备融合。融合后，达到最终的 6 螺旋束 (6HB) 构象涉及形成对称 6HB 三聚体所需的分子间相互作用与 gp41 胞外域（包括其近膜区域）的膜亲和力之间的竞争。我们对包含 gp41 胞外域（带或不带近膜区域）的三种构建体的结构和动态特性进行的溶液 NMR 研究表明，这些片段直到融合过程的最后步骤才形成螺旋间相互作用。因此，不与脂质表面缔合的七肽区域的极性末端之间的相互作用可能构成引发最终融合后状态形成的主要驱动力。在存在十二烷基磷酸胆碱和由 DMPC 和二己酰磷脂酰胆碱组成的 bicelles 的情况下，不存在显着的分子间胞外域相互作用，这表明 gp41s 跨膜螺旋三聚化对于防止融合过程中三聚体完全解离的重要性。 HIV-1 gp41 的膜近端外部区域 (MPER) 包含至少四种广泛中和抗体的表位。根据解决方案条件和构造设计，已经报道了该部分的不同结构。我们发现，在水溶液中，MPER 片段（gp160 残基 660-674）以单体-三聚体平衡存在，缔合常数在微摩尔范围内。热力学分析表明，这种缔合是放热的，在 D2O 中比在 H2O 中更有利，并且随着离子强度的增加而增加，表明疏水性驱动的分子间相互作用。圆二色性、13C 化学位移、NOE 和氢交换率表明，MPER 经历了从低浓度下以未折叠为主的单体到高浓度下的 α 螺旋三聚体的结构转变。该结果对于 gp41 从发夹前中间体转变为其融合后 6 螺旋束状态的过程之前和过程中 MPER 的抗体识别具有影响。 应用于 SARS-CoV-2 刺突蛋白胞外域的初步实验表明，该蛋白含有一个三螺旋 N 端七肽重复序列 (NHR)，比 gp41 的相应区域稳定得多，并且倾向于C 端七肽 (CHR) 重复序列虽然低于 NHR，但也显着高于 gp41 的 CHR，而其膜近端区域三聚化的倾向大大降低。 作品已提交出版，但仍在审查中。