Park Sang Ho, Mrse Anthony A, Nevzorov Alexander A, Mesleh Michael F, Oblatt-Montal Myrta, Montal Mauricio, Opella Stanley J
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0307, USA.
J Mol Biol. 2003 Oct 17;333(2):409-24. doi: 10.1016/j.jmb.2003.08.048.
The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu(2-30+) is a 36-residue polypeptide that consists of residues 2-30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu(2-30+) in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu(2-30+) in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees for residues 17-25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 A. Vpu(2-30+) exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(+/- 6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.
通过核磁共振波谱法在胶束和双层样品中测定了HIV-1病毒蛋白“u”（Vpu）的通道形成跨膜结构域的三维结构。Vpu(2 - 30+)是一种由36个残基组成的多肽，它包含Vpu N端的2 - 30位残基以及C端的一个六残基“溶解性标签”，该标签有助于分离、纯化和制备这个高度疏水的最小通道形成结构域。在胶束中均匀15N标记的Vpu(2 - 30+)的二维1H/15N HSQC谱中，几乎所有的共振峰都与全长Vpu谱中相应残基的共振峰重叠，这表明跨膜结构域的结构不受其C端胞质结构域存在的强烈影响。Vpu(2 - 30+)在玻璃板之间排列的脂质双层中的二维1H/15N PISEMA谱已完全解析并归属。谱中的“轮状”共振模式是轻微倾斜的跨膜螺旋的特征。还对弱排列的胶束样品进行了实验，以测量残余偶极耦合和化学位移各向异性。对从弱排列和完全排列样品获得的PISA轮和偶极波的分析表明，Vpu(2 - 30+)有一个跨膜α螺旋，跨越8 - 25位残基，平均倾斜度为13度。该螺旋在Ile17处略有弯曲，导致8 - 16位残基的倾斜度为12度，17 - 25位残基的倾斜度为15度。与实验固态核磁共振数据的结构拟合得到了一个精度相当于均方根偏差为0.4 Å的三维结构。Vpu(2 - 30+)在PFO - PAGE上主要以寡聚体形式存在，并形成离子通道，在脂质双层中的最常见电导率为96(±6) pS。跨膜结构域的结构特征是离子通道活性的决定因素，这可能与该蛋白在促进新病毒颗粒从感染细胞中出芽的作用有关。
