Bentrop D, Bertini I, Cremonini M A, Forsén S, Luchinat C, Malmendal A
Department of Chemistry, University of Florence, Via Gino Capponi 7, 50121 Florence, Italy.
Biochemistry. 1997 Sep 30;36(39):11605-18. doi: 10.1021/bi971022+.
The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.
利用铈离子（Ce3+）引入的顺磁T1弛豫增强和赝接触位移，结合传统的NOE约束条件，解析了钙调蛋白N端结构域的二铈（III）配合物（以下简称Ce2-TR1C）的溶液结构。使用赝接触位移约束条件是通过核磁共振（NMR）在蛋白质结构中定位金属离子的首次尝试。与钙（II）类似，已发现顺磁铈（III）以协同方式结合到钙调蛋白TR1C片段的两个金属结合位点上。由于Ce3+离子与76个残基的蛋白质中的质子之间存在赝接触相互作用，该配合物的1H NMR谱显示共振峰在+22至-9 ppm之间移动。通过使用TOCSY/COSY和NOESY谱的标准方法，以及针对靠近顺磁离子的质子的宽共振峰的一维NOE差谱，可以归属其80%的质子共振峰。借助扭转角动力学程序DYANA [Güntert, P., Mumenthaler, C., & Wüthrich, K. (1996) XVIIth International Conference on Magnetic Resonance in Biological Systems (Abstract)]，利用1012个NOE计算出了一系列结构。纵向质子弛豫时间有助于大致确定金属离子在蛋白质中的位置。随后添加了总共381个赝接触位移约束条件（对其评估和使用进行了严格讨论），以进一步优化蛋白质框架内的金属坐标并提高结构分辨率。观察到金属坐标的分辨率有显著提高，并且在靠近顺磁中心、NOE数量较少的区域，分辨率也有相当大的提高。溶液结构的良好质量使得能够与分辨率为1.7 Å的钙负载钙调蛋白的固态结构进行有意义的比较[Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]。Ce2-TR1C配合物总体上比钙形式更紧凑。
