Structural study of the HIV1 gp41 coat protein

HIV1 gp41外壳蛋白的结构研究

• 批准号: 8939688
• 负责人: Ad Bax
• 金额: $ 77.46万
• 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/8939688
• 关键词: Adopted; C-terminal; Capsid Proteins; Data; Detergents; Diffusion; Disease; Equilibrium; Exhibits; Flu virus; HIV-1; Hemagglutinin; Homo; Left; Mediating; Membrane; Membrane Fusion; Micelles; Modeling; Molecular Conformation; Peptides; Phospholipids; Property; Proteins; Relaxation; Reporting; Role; Solutions; Staging; Structure; Temperature; Time; Transmembrane Domain; Virus; falls; immunogenic; light scattering; protein aggregate; stoichiometry; unilamellar vesicle

Truncation of the gp41 construct just past its transmembrane domain, leaving residues 1-194, results in a protein that is soluble in detergent micelles. Although previously believed to adopt its natural homo-trimeric form, we have found strong evidence for a rapid dynamic equilibrium between monomeric and trimeric forms of the protein, which can be shifted to the mostly trimeric form by choice of suitable detergents and detergent:protein stoichiometry. The NOE data and 15N relaxation rates show a well structured helical conformation for residues 5-14 of the fusion peptide region, followed by a more disordered FPPR segment which connects it to the ecto-domain. Remarkably, resonances from the C-terminal heptad repeat (CHR) and membrane proximal region MPER) are exchange broadened to such an extent that they do not give rise to observable NMR resonances. Together with exchange broadening observed in the highly mobile immuno-dominant loop region, this points to an equilibrium between early and late stage 3- and 6-helical states for the ecto domain. There is no evidence for direct interaction with the transmembrane fusion domain in our studies carried out at pH4, and both light scattering, SAXS, and NMR diffusion data point to a mass ratio of detergent:protein slightly above 1.0. The protein is found to be quite stable, even at temperatures of 40C. The fusion peptide exhibits effective correlation times that are much shorter than for the ecto domain, indicating that the intact fusion domain helix is highly mobile within the detergent micelle/protein aggregate, while retaining a helical conformation. This excludes the previously hypothesized interaction with the gp41 transmembrane domain under our conditions of pH and detergent. When studying a protein construct that consists of just the N- and C-terminal heptad repeat regions of gp41 (NHR and CHR), with the immunogenic loop region replaces by a short linker, our NMR data indicate the 6-helical bundle structure adopted by the protein is indistinguishable from the crystallographic model reported for this structure. However, we find that in the presence of detergent micelles or small unilamellar vesicles, the trimer falls apart while the NHR and CHR retain their alpha-helical structure. Both helices were found to interact tightly with the phospholipids, suggesting an active role for the gp41 ecto domain in mediating membrane fusion.

gp 41构建体的截短刚好超过其跨膜结构域，留下残基1-194，产生可溶于洗涤剂胶束的蛋白质。 虽然先前认为采用其天然的同源三聚体形式，但我们已经发现了蛋白质的单体和三聚体形式之间快速动态平衡的有力证据，通过选择合适的去污剂和去污剂：蛋白质化学计量，可以将其转变为主要的三聚体形式。NOE数据和15 N弛豫速率显示融合肽区的残基5-14具有良好结构的螺旋构象，随后是将其连接至胞外结构域的更无序的FPPR区段。 值得注意的是，来自C-末端七肽重复序列（HPR）和膜近端区域（MPER）的共振被交换加宽到这样的程度，使得它们不产生可观察到的NMR共振。 连同在高度移动的免疫显性环区域中观察到的交换增宽，这指向了外结构域的早期和晚期3-和6-螺旋状态之间的平衡。 在我们的研究中，在pH 4下进行的跨膜融合结构域没有直接相互作用的证据，光散射、SAXS和NMR扩散数据都指向去污剂：蛋白质的质量比略高于1.0。这种蛋白质即使在40 ℃的温度下也相当稳定。 融合肽显示出比胞外结构域短得多的有效相关时间，表明完整的融合结构域螺旋在去污剂胶束/蛋白质聚集体内是高度移动的，同时保留螺旋构象。这排除了先前假设的在我们的pH和去污剂条件下与gp 41跨膜结构域的相互作用。当研究仅由gp 41的N-和C-末端七肽重复区域（NHR和NHR）组成的蛋白质构建体时，免疫原性环区域被短接头取代，我们的NMR数据表明蛋白质采用的6-螺旋束结构与针对该结构报道的晶体学模型无法区分。 然而，我们发现，在洗涤剂胶束或小单层囊泡的存在下，三聚体福尔斯脱落，而NHR和NHR保留其α-螺旋结构。 这两个螺旋被发现与磷脂紧密相互作用，表明gp 41胞外结构域在介导膜融合中的积极作用。