Skelton N J, Kördel J, Chazin W J
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
J Mol Biol. 1995 Jun 2;249(2):441-62. doi: 10.1006/jmbi.1995.0308.
The three-dimensional structure of apo calbindin D9k has been determined using constraints generated from nuclear magnetic resonance spectroscopy. The family of solution structures was calculated using a combination of distance geometry, restrained molecular dynamics, and hybrid relaxation matrix analysis of the nuclear Overhauser effect (NOE) cross-peak intensities. Errors and inconsistencies in the input constraints were identified using complete relaxation matrix analyses based on the results of preliminary structure calculations. The final input data consisted of 994 NOE distance constraints and 122 dihedral constraints, aided by the stereospecific assignment of the resonances from 21 beta-methylene groups and seven isopropyl groups of leucine and valine residues. The resulting family of 33 structures contain no violation of the distance constraints greater than 0.17 A or of the dihedral angle constraints greater than 10 degrees. The structures consist of a well-defined, antiparallel four-helix bundle, with a short anti-parallel beta-interaction between the two unoccupied calcium-binding loops. The root-mean-square deviation from the mean structure of the backbone heavy-atoms for the well-defined helical residues is 0.55 A. The remainder of the ion-binding loops, the linker loop connecting the two sub-domains of the protein, and the N and C termini exhibit considerable disorder between different structures in the ensemble. A comparison with the structure of the (Ca2+)2 state indicates that the largest changes associated with ion-binding occur in the middle of helix IV and in the packing of helix III onto the remainder of the protein. The change in conformation of these helices is associated with a subtle reorganization of many residues in the hydrophobic core, including some side-chains that are up to 15 A from the ion-binding site.
已使用核磁共振光谱产生的约束条件确定了脱钙钙结合蛋白D9k的三维结构。通过距离几何、受限分子动力学以及对核Overhauser效应（NOE）交叉峰强度的混合弛豫矩阵分析相结合的方法，计算了溶液结构家族。基于初步结构计算的结果，使用完全弛豫矩阵分析确定了输入约束中的误差和不一致性。最终的输入数据包括994个NOE距离约束和122个二面角约束，21个β-亚甲基基团以及亮氨酸和缬氨酸残基的7个异丙基的共振立体专一性归属对其有辅助作用。由此产生的33个结构的家族中，没有违反大于0.17 Å的距离约束或大于10度的二面角约束。这些结构由一个定义明确的反平行四螺旋束组成，在两个未占据的钙结合环之间有一个短的反平行β相互作用。对于定义明确的螺旋残基，其主链重原子相对于平均结构的均方根偏差为0.55 Å。离子结合环的其余部分、连接蛋白质两个亚结构域的连接环以及N和C末端在整体的不同结构之间表现出相当大的无序性。与（Ca2+）2状态的结构比较表明，与离子结合相关的最大变化发生在螺旋IV的中部以及螺旋III与蛋白质其余部分的堆积中。这些螺旋构象的变化与疏水核心中许多残基的细微重组有关，包括一些距离离子结合位点达15 Å的侧链。
