Sarzedas Carolina G, Lima Carla S, Juliano Maria A, Juliano Luiz, Valente Ana Paula, Da Poian Andrea T, Almeida Fabio C L
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.
J Pept Sci. 2008 Apr;14(4):429-35. doi: 10.1002/psc.934.
Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].
有包膜动物病毒进入宿主细胞总是依赖于病毒包膜糖蛋白构象变化引发的膜融合步骤。水疱性口炎病毒（VSV）感染由病毒刺突糖蛋白G介导，该蛋白在内体区室的酸性环境中诱导膜融合。在之前的工作中，我们在VSV G蛋白中鉴定出一个特定序列，包含145 - 164位残基，直接参与膜相互作用和融合。在本工作中，我们使用核磁共振研究了肽段pep[145 - 164]与膜的相互作用，以解析该肽段在两种膜模拟体系中的结构：SDS胶束以及由磷脂酰胆碱和磷脂酰丝氨酸组成的脂质体（PC:PS囊泡）。中程核Overhauser效应（NOE）的存在表明，在SDS胶束存在的情况下，该肽段倾向于形成N端和C端螺旋片段。化学位移指数分析表明，在所研究的所有条件下，C端螺旋处于螺旋 - 无规卷曲平衡状态。在pH 7.0时，当pep[145 - 164]在溶液中游离或存在PC:PS时，N端螺旋也呈现螺旋 - 无规卷曲平衡。值得注意的是，在融合pH值下，在SDS或PC:PS存在时，N端螺旋区域呈现出与螺旋和无规卷曲处于平衡状态的第三种构象形式。N端螺旋含量随pH降低，并且在融合pH值下，次要的β结构构象变得更为普遍。这些数据表明β构象是融合活性结构，这与X射线结构一致，X射线结构显示对应于pep[145 - 164]区域的β发夹结构。
