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.

GP41构建体的截断刚刚超过了其跨膜结构域，使残基1-194留下，导致蛋白质可溶于洗涤剂胶束。 尽管以前据信采用其天然均匀形式，但我们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，可以通过选择合适的洗涤剂和清洁剂：蛋白质stoichiimentry将其转移到三聚体形式。 NOE数据和15N弛豫率显示了融合肽区域5-14的残基的结构良好的螺旋构象，然后是更无序的FPPR段，将其连接到ecto域。 值得注意的是，C末端七体重复（CHR）和膜近端区域MPER的共振得到了扩展，以至于它们不会引起可观察到的NMR共振。 加上在高度流动免疫主导的环路中观察到的交换扩大，这表明了ECTO域的早期和晚期3-螺旋状态之间的平衡。 在我们在PH4上进行的研究中，没有证据表明与跨膜融合结构域的直接相互作用，并且光散射，SAXS和NMR扩散数据都表明了洗涤剂的质量比：蛋白质略高于1.0。即使在40℃的温度下，该蛋白质也很稳定。 融合肽表现出比ECTO结构域短得多的有效相关时间，这表明完整的融合结构域螺旋在洗涤剂胶束/蛋白质聚集体中高度流动，同时保留了螺旋构象。这排除了先前假设的与GP41跨膜结构域的相互作用，在我们的pH和洗涤剂条件下。当研究仅由GP41（NHR和CHR）的N和C端重复区域组成的蛋白质构建体时，免疫原性环区域替代了一个短接头，我们的NMR数据表明，该蛋白质与蛋白质所采用的6螺旋束结构与该结构的晶体模型无关。 但是，我们发现在存在洗涤剂胶束或小的单层囊泡的情况下，三聚体散落，而NHR和CHR保留其α-螺旋结构。 发现这两个螺旋都与磷脂紧密相互作用，这表明GP41 ECTO结构域在介导膜融合中起着活性作用。