Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1
Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1
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DOI：
10.1016/j.jmb.2003.08.048
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发表时间：
2003-10-17
影响因子：
5.6
通讯作者：
Opella, SJ
中科院分区：
文献类型：
--
作者：
Park, SH;Mrse, AA;Opella, SJ
用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）通道形成跨膜结构域的三维结构。Vpu（2-30+）是36个残基的多肽，其由Vpu的N末端的残基2-30和在其C末端的六个残基的“溶解性标签”组成，所述溶解性标签促进该高度疏水的最小通道形成结构域的分离、纯化和样品制备。胶束中均匀N-15标记的Vpu（2-30+）的二维H-1/N-15 HSQC光谱中的几乎所有共振都可叠加在全长Vpu光谱中相应残基的共振上，这表明跨膜结构域的结构不受其C末端胞质结构域的存在的强烈影响。本文对玻璃板间排列的脂双层中Vpu（2-30+）的H ~（-1）/N ~（-15）二维PISEMA谱进行了完全分辨和归属。光谱中的“轮状”共振模式是略微倾斜的跨膜螺旋的特征。实验还进行了弱对齐胶束样品，以测量残余偶极耦合和化学位移各向异性。从弱排列和完全排列的样品获得的比萨轮和偶极波的分析表明，Vpu（2-30+）具有跨膜α-螺旋，跨越残基8-25，平均倾斜13度。螺旋在Ile 17处轻微扭结，这导致残基8-16倾斜12度，残基17-25倾斜15度。与实验固态NMR数据的结构拟合导致具有相当于0.4埃的RMSD的精度的三维结构。Vpu（2-30+）在PFO-PAGE上主要以寡聚体形式存在，并形成离子通道，在脂质双层中最常见的电导率为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，离子通道活性可能与蛋白质在促进新病毒颗粒从感染细胞出芽中的作用有关。(C)2003 Elsevier Ltd.保留所有权利。
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 H-1/N-15 HSQC spectrum of uniformly N-15 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 H-1/N-15 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 13degrees. The helix is kinked slightly at Ile17, which results in tilts of 12degrees for residues 8-16 and 15degrees 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 Angstrom. 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. (C) 2003 Elsevier Ltd. All rights reserved.