Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion.

Erns是一种具备核糖核酸酶（RNase）活性的病毒粒子必需糖蛋白，当其从受感染细胞中部分分泌后，可抑制宿主细胞的天然免疫应答。其非同寻常的C端具有多重功能：两亲性螺旋可同时充当膜锚定结构、信号肽酶切割位点以及驻留/分泌信号。为更好地解析其功能背后的潜在分子机制，本研究对该序列的结构与膜结合特性展开了分析。不同实验体系下的圆二色光谱（CD spectroscopy）检测，结合蒙特卡洛（Monte Carlo）模拟与分子动力学模拟（molecular dynamics simulations），证实了该序列的螺旋折叠特性，并揭示该螺旋以轻微倾斜的方式嵌入脂质双层的两亲性区域，而非与膜表面平行排布。该结构模型通过核磁共振（NMR）分析得到了验证，同时核磁共振分析还鉴定出该螺旋中央存在一段由15个氨基酸残基组成的区域，该区域完全被水相所屏蔽，其C端紧邻一段推定的发夹结构。上述研究结果阐明了该蛋白具备强膜结合能力的分子基础，并为解析Erns的膜接触、加工及分泌过程提供了关键线索。