Structural study of the HIV1 gp41 coat protein

HIV1 gp41外壳蛋白的结构研究

• 批准号: 8553623
• 负责人: Ad Bax
• 金额: $ 30.06万
• 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/8553623
• 关键词: Adopted; C-terminal; Capsid Proteins; Data; Detergents; Diffusion; Disease; Equilibrium; Exhibits; Flu virus; HIV-1; Hemagglutinin; Homo; Left; Membrane; Micelles; Molecular Conformation; Property; Proteins; Relaxation; Solutions; Staging; Structure; Temperature; Time; Transmembrane Domain; Virus; light scattering; protein aggregate; protein structure; stoichiometry

Truncation of the gp41 construct just past its transmembrane domain, leaving residues 1-194, results in a well-structured protein, 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 domain, followed by a more disordered 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 a possible 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 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 domain exhibits effective correlation times that are much shorter than for the ecto domain, indicating that the intact fusion domain helices are highly mobile within the detergent micelle/protein aggregate, while retaining their helical conformation. This excludes the previously hypothesized interaction with the gp41 transmembrane domain under our conditions of pH and detergent.

GP41构建体的截断刚刚超过其跨膜结构域，留下了1-194的残基导致结构良好的蛋白质，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弛豫率显示了融合域5-14的残基的结构良好的螺旋构象，其次是更无序的节段，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。 加上在高度移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-螺旋状态之间可能的平衡。 在PH4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，光散射，SAXS和NMR扩散数据都表明洗涤剂的质量比：蛋白质略高于1.0。即使在40℃的温度下，该蛋白质也很稳定。 融合结构域的有效相关时间比ECTO结构域短得多，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，同时保留其螺旋构象。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。