Rabenstein M, Shin Y K
Department of Chemistry, University of California, Berkeley 94720, USA.
Biochemistry. 1995 Oct 17;34(41):13390-7. doi: 10.1021/bi00041a016.
The envelope glycoprotein gp41 from human immunodeficiency virus type 1 (HIV-1) is involved in membrane fusion and virus entry. It contains a functionally important leucine zipper-like heptad repeat region (residues 553-590). To investigate the solution structure and membrane-binding properties of this region, cysteine-substituted variants of a 38-residue peptide derived from the heptad repeat were synthesized and modified with nitroxide spin labels. Analytical equilibrium ultracentrifugation studies indicated it is primarily tetrameric in solution, in contrast to the protein gp160 which is a mixture of trimers and tetramers. Electron paramagnetic resonance (EPR) measurements indicated that the peptide was bound to vesicles containing 10 mol % negatively charged lipids. The peptides were bound parallel to the membrane surface, near the water-membrane interface, in a structure different from the solution structure, most likely as monomers. When Asp, Pro, or Ser was substituted for Ile at the core "a" position of the heptad repeat in the middle of the peptide, the coiled coil was destabilized. In addition, these peptides showed reduced membrane-binding affinities. Thus, mutations that destabilized coiled-coil formation also decreased membrane-binding propensity. These experimental results, taken with previous evidence, suggest two functions for the heptad repeat of gp41 after CD4 binding: (1) to form an extended coiled coil; (2) to provide a hydrophobic face that binds to the host-cell membrane, bringing the viral and cellular membranes closer and facilitating fusion.
人类免疫缺陷病毒1型（HIV-1）的包膜糖蛋白gp41参与膜融合和病毒进入过程。它包含一个功能上重要的亮氨酸拉链样七肽重复区域（残基553 - 590）。为了研究该区域的溶液结构和膜结合特性，合成了源自七肽重复序列的38个残基肽的半胱氨酸取代变体，并用氮氧自旋标记进行修饰。分析型平衡超速离心研究表明，与三聚体和四聚体混合物的蛋白gp160不同，该肽在溶液中主要以四聚体形式存在。电子顺磁共振（EPR）测量表明，该肽与含有10摩尔%带负电荷脂质的囊泡结合。这些肽平行于膜表面，靠近水 - 膜界面结合，其结构与溶液结构不同，很可能以单体形式存在。当在肽中间的七肽重复序列的核心“a”位置用天冬氨酸、脯氨酸或丝氨酸取代异亮氨酸时，卷曲螺旋结构不稳定。此外，这些肽表现出降低的膜结合亲和力。因此，破坏卷曲螺旋形成的突变也会降低膜结合倾向。这些实验结果与先前的证据一起表明，gp41的七肽重复序列在CD4结合后具有两种功能：（1）形成延伸的卷曲螺旋；（2）提供一个与宿主细胞膜结合的疏水表面，使病毒膜和细胞膜更接近并促进融合。
