Korazim Ofir, Sackett Kelly, Shai Yechiel
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel.
J Mol Biol. 2006 Dec 15;364(5):1103-17. doi: 10.1016/j.jmb.2006.08.091. Epub 2006 Sep 5.
HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from alpha-helix in an outer leaflet-like zwitterionic membrane to beta-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation. We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion inhibitors inhibit HIV entry in an analogous mechanism.
HIV-1进入宿主细胞涉及一系列相互作用，其中gp41与质膜直接接触。了解膜组成对融合机制的影响可以揭示这一复杂机制中尚未解决的阶段。在这里，我们研究了N36，一种源自gp41胞外域N-七肽重复序列（NHR）的肽，其形成六螺旋束（SHB）的对应物C34，以及N端70聚体野生型肽（N70），还有在两种模拟质膜内外小叶的膜存在下的其他gp41胞外域衍生肽。通过各种荧光、光谱和显微镜方法收集了这些肽的结构信息、它们对磷脂的亲和力以及它们诱导囊泡融合的能力。我们发现，对磷脂具有强亲和力的N36在类似外小叶的两性离子膜中从α-螺旋显著转变为在模拟带负电荷的内小叶环境的膜中的β-折叠，从而导致明显的融合活性。实时原子力显微镜（AFM）用于研究肽对膜形态的影响，揭示了严重的双层扰动和广泛的孔形成。我们还发现，N36/C34核心被带负电而非两性离子的磷脂破坏稳定性。综上所述，我们的数据表明，gp41的融合活性孔形成构象在SHB的上游延伸。通过这种方式，胞外域折叠成SHB也可能通过阻碍gp41的融合活性表面作为融合的负调节剂，从而防止对细胞膜的不可逆损伤。这一假设得到以下发现的支持：用C-七肽重复序列（CHR）衍生的融合抑制剂对大单层囊泡（LUV）进行预孵育以剂量依赖的方式降低N端肽的融合活性，并表明CHR衍生的融合抑制剂以类似机制抑制HIV进入。
