Federau T, Schubert U, Flossdorf J, Henklein P, Schomburg D, Wray V
Abteilung für Molekulare Strukturforschung, GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
Int J Pept Protein Res. 1996 Apr;47(4):297-310. doi: 10.1111/j.1399-3011.1996.tb01359.x.
The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.
HIV-1特异性Vpu蛋白是一种由81个氨基酸组成的I类整合膜磷蛋白，它在内质网中诱导病毒受体CD4的降解，并增强病毒颗粒从感染细胞中的释放。Vpu具有两亲性，由靠近极性C末端胞质结构域的疏水N末端膜锚定区组成。在我们最近专注于胞质尾巴结构分析的工作中，我们建立了一个α-螺旋-柔性-α-螺旋-转角模型。现在我们展示了Vpu胞质结构域的实验溶液结构，该结构已通过二维1H NMR光谱、受限分子动力学和能量最小化计算在50%三氟乙醇水溶液中阐明。在这些条件下，肽Vpu32-81主要为单体形式，并采用明确的螺旋-连接-螺旋-转角构象，其中四个区域由残基37-51、52-56、57-72和73-78界定。Pro-75顺式异构体的存在表现为二维光谱中相邻残基交叉峰的加倍。一种相关的变体肽Vpum32-81，其中Vpu磷酸化位点Ser52和Ser56被替换为Asn，采用非常相似的结构，总体而言，这证明第二个螺旋和转角形成了一个相对刚性的区域。两个螺旋都具有两亲性，但电荷分布不同。一般来说，胞质区域在N末端带正电荷，在螺旋1中穿过一个电荷交替的区域，然后变为带负电荷。连接区的灵活性允许两个螺旋有取向自由度。这里发现的基序是Vpu胞质结构域的第一个通过实验精制的溶液结构，可以想象这些α-螺旋对于与位于CD4胞质尾巴内的α-螺旋Vpu反应元件先前定义的物理相互作用很重要。
