Structural study of the HIV1 gp41 coat protein

gp41结构体的截短刚好经过其跨膜结构域，留下残基1-194，产生可溶于洗涤剂胶束的蛋白质。虽然以前认为采用天然的三聚体形式，但我们已经发现了强有力的证据，证明蛋白质的单体和三聚体形式之间的快速动态平衡，可以通过选择合适的洗涤剂和洗涤剂转移到主要的三聚体形式：蛋白质化学计量学。NOE数据和15N弛豫率显示融合肽区域残基5-14具有结构良好的螺旋构象，随后是一个更无序的FPPR片段，将其连接到外结构域。值得注意的是，来自c端七重体（CHR）和膜近端区域MPER)的共振被交换拓宽到这样的程度，以至于它们不会产生可观察到的核磁共振共振。加上在高度移动的免疫优势环区观察到的交换展宽，这表明在ecto结构域的早期和晚期阶段3-和6-螺旋状态之间存在平衡。在我们在pH4下进行的研究中，没有证据表明与跨膜融合域直接相互作用，光散射、SAXS和NMR扩散数据都指向洗涤剂：蛋白质的质量比略高于1.0。人们发现，即使在40摄氏度的温度下，这种蛋白质也相当稳定。融合肽显示出比外链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/蛋白质聚集体中具有高度的流动性，同时保留了螺旋构象。这排除了先前假设的在我们的pH和洗涤剂条件下与gp41跨膜结构域的相互作用。当研究gp41 （NHR和CHR）的N端和c端七端重复区组成的蛋白质结构时，我们的核磁共振数据表明，蛋白质采用的6螺旋束结构与该结构的晶体学模型无法区分。然而，我们发现，在洗涤剂胶束或小的单层囊泡的存在下，三聚体分崩离析，而NHR和CHR保持其α螺旋结构。这两个螺旋被发现与磷脂紧密相互作用，表明gp41外显子结构域在介导膜融合中起积极作用。

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.