Lin Ta-Hsien, Chia Chih-Ming, Hsiao Jye-Chian, Chang Wen, Ku Chiao-Chu, Hung Shang-Cheng, Tzou Der-Lii M
Institute of Biochemistry, National Yang-Ming University, Shih-pai, Taipei 11221, Taiwan.
J Biol Chem. 2002 Jun 7;277(23):20949-59. doi: 10.1074/jbc.M110403200. Epub 2002 Mar 18.
This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid alpha-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid alpha-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual alpha-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu(41), Leu(45), and Leu(48)) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.
本研究展示了通过核磁共振（NMR）和圆二色光谱（CD）确定的痘苗病毒包膜蛋白A27L胞外结构域的分子结构。构建了一种重组蛋白eA27L-aa，其包含该结构域，其中第71和72位半胱氨酸被丙氨酸取代，以防止由于这两个半胱氨酸之间的分子间二硫键导致的自组装。可溶性eA27L-aa蛋白形成一种类似于痘苗病毒粒子上A27L的寡聚体。在有或没有尿素存在的情况下，对eA27L-aa进行了异核相关NMR光谱分析，以确定主链共振归属。化学位移指数（CSI）倾向分析表明，eA27L-aa有两个不同的结构域，N端区域有一个相对灵活的22个氨基酸的无规卷曲，结构其余部分有一个相当刚性的α-螺旋结构。使用等温滴定量热法进行的结合相互作用研究表明，N端区域一个富含12个氨基酸的赖氨酸/精氨酸片段负责与糖胺聚糖结合。eA27L-aa的刚性α-螺旋部分可能参与内在自组装，CSI倾向分析表明，具有残余α-螺旋倾向的N37-E49区域可能是自组装核心。自组装归因于该片段中的三个疏水性亮氨酸残基（Leu(41)、Leu(45)和Leu(48)）。通过CD光谱分析了eA27L-aa的折叠机制，结果显示在没有尿素的情况下有一个两步转变，吉布斯自由能为2.5千卡/摩尔。基于这些NMR和CD研究，提出了A27L胞外结构域的残基特异性分子模型。这些关于eA27L-aa分子结构的研究将有助于理解痘苗病毒如何进入细胞。
