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.

gp 41构建体的截短刚好超过其跨膜结构域，留下残基1-194，导致结构良好的蛋白质，可溶于洗涤剂胶束。 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弛豫速率显示融合结构域的残基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片段。 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，这表明对于胞外结构域，早期和晚期3-和6-螺旋状态之间可能存在平衡。 在pH 4下进行的研究中，没有证据表明与跨膜融合结构域直接相互作用，光散射、SAXS和NMR扩散数据都表明去污剂：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。 融合结构域表现出有效的相关时间比外结构域短得多，表明完整的融合结构域螺旋在洗涤剂胶束/蛋白质聚集体内是高度移动的，同时保留其螺旋构象。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。