Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy.
Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy.
复制标题
通过异核三维和四维核磁共振波谱测定 ras p21.GDP 的溶液结构和动力学。
作者：
Per;J.;Kraulis;Peter;Domaille;Sharon L. Campbell;Thomas Van Aken;Ernest D. Laue’J
使用核磁共振波谱法测定了 ras p21 蛋白截短版本（残基 1-166）的 GDP 形式的高分辨率溶液结构。 Ras p21 是人类 ras 原癌基因的产物，是普遍存在的真核基因家族的成员，该基因家族在进化中高度保守。使用新开发的 3D/4D 版本 ANSIG 软件分析三维和四维（3D 和 4D）异核 NMR 谱，获得了几乎完整的归属（13C、15N 和 1H），包括缬氨酸残基的 54 C β 亚甲基质子和 10 C γ 甲基质子的立体特异性归属。根据 3369 个实验约束计算出总共 40 个收敛结构，其中包括 3,167 个核欧沃豪塞效应 (NOE) 衍生距离、14 phi 和 54 chi 1 扭转角约束、109 个氢键距离约束以及源自定义 GDP 配体、镁离子和蛋白质之间相互作用的文献数据的另外 25 个约束。残基 58-66（环 L4）以及残基 30-38（环 L2）区域的结构不明确。对蛋白质主链 15N 核的动力学分析表明，58-66、107-109 区域内的残基以及较小程度的 30-38 区域内的残基在纳秒时间尺度上动态移动。如果分析中包含所有残基 (1-166)，则 40 个溶液结构与平均原子坐标之间的均方根 (rms) 偏差对于主链重原子为 0.78 A，对于所有非氢原子为 1.29 A。如果从分析中排除残基 30-38 和残基 58-66，则 rms 偏差将分别降至 0.55 A 和 1.00 A。该结构与 ras p21.GDP (1-189) 最精细的 X 射线晶体结构进行了比较 [Milburn, M. V., Tong, L., de Vos, A. M., Brünger, A. T., Yamaizumi, Z., Nishimura, S., & Kim, S.-H. (1990) 科学 24, 939-945]。除了通过核磁共振波谱发现可移动的区域外，结构非常相似。此外，第二个 α 螺旋（helix-2）的方向略有不同。如果从分析中排除残基 31-37 和残基 59-73，则主链重原子的溶液结构平均值与 X 射线晶体结构之间的 rms 偏差为 0.94 A。
A high-resolution solution structure of the GDP form of a truncated version of the ras p21 protein (residues 1-166) has been determined using NMR spectroscopy. Ras p21 is the product of the human ras protooncogene and a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. A virtually complete assignment (13C, 15N, and 1H), including stereospecific assignments of 54 C beta methylene protons and 10 C gamma methyl protons of valine residues, was obtained by analysis of three- and four-dimensional (3D and 4D) heteronuclear NMR spectra using a newly developed 3D/4D version of the ANSIG software. A total of 40 converged structures were computed from 3369 experimental restraints consisting of 3,167 nuclear Overhauser effect (NOE) derived distances, 14 phi and 54 chi 1 torsion angle restraints, 109 hydrogen bond distance restraints, and an additional 25 restraints derived from literature data defining interactions between the GDP ligand, the magnesium ion, and the protein. The structure in the region of residues 58-66 (loop L4), and to a lesser degree residues 30-38 (loop L2), is ill-defined. Analysis of the dynamics of the backbone 15N nuclei in the protein showed that residues within the regions 58-66, 107-109, and, to a lesser degree, 30-38 are dynamically mobile on the nanosecond time scale. The root mean square (rms) deviations between the 40 solution structures and the mean atomic coordinates are 0.78 A for the backbone heavy atoms and 1.29 A for all non-hydrogen atoms if all residues (1-166) are included in the analysis. If residues 30-38 and residues 58-66 are excluded from the analysis, the rms deviations are reduced to 0.55 and 1.00 A, respectively. The structure was compared to the most highly refined X-ray crystal structure of ras p21.GDP (1-189) [Milburn, M. V., Tong, L., de Vos, A. M., Brünger, A. T., Yamaizumi, Z., Nishimura, S., & Kim, S.-H. (1990) Science 24, 939-945]. The structures are very similar except in the regions found to be mobile by NMR spectroscopy. In addition, the second alpha-helix (helix-2) has a slightly different orientation. The rms deviation between the average of the solution structures and the X-ray crystal structure is 0.94 A for the backbone heavy atoms if residues 31-37 and residues 59-73 are excluded from the analysis.