Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution.
Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution.
复制标题
核糖核酸酶 T1 与鸟苷基-2,5-鸟苷复合的三维结构，分辨率为 1.8 A。
DOI：
10.1016/0022-2836(89)90495-6
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发表时间：
1989
影响因子：
5.6
通讯作者：
W. Saenger
中科院分区：
文献类型：
--
作者：
J. Koepke;M. Maslowska;U. Heinemann;W. Saenger
从黑曲霉中分离的核糖核酸酶T1（RNase T1）与特异性抑制剂鸟苷酰-2 ′，5′-鸟苷（2′，5′-GpG）共结晶，用立体化学限制最小二乘精修法对结构进行精修，在6 - 1.8 μ m分辨率范围内，X射线衍射数据的晶体学R因子为14.9%。改进的模型由781个蛋白质原子，43个抑制剂原子在一个主要网站和29个抑制剂原子在一个次要网站，107个水氧原子，和一个金属网站指定为钙。在最后的改进，His，Asn和Gln侧链的取向重新解释的基础上的二维核磁共振数据。RNase T 1/2′，5′-GpG复合物和近同晶型RNase T 1/2′-GMP复合物的晶体堆积和酶构象是可比的。等效蛋白质原子之间的均方根偏差为0.73 μ m。晶胞尺寸的差异主要是由于结合的抑制剂。2′，5′-GpG的5′-末端鸟嘌呤与RNase t1的结合方式与2′-GMP复合物中的结合方式大致相同。相比之下，催化中心与磷酸基团之间的氢键不同，3 '端鸟嘌呤不与酶形成氢键。这种差的结合反映在2′，5′-GpG（5′-末端鸟嘌呤除外）的2倍紊乱中，这源于5′-末端核糖的褶皱差异。主要位点的褶皱为C2′-exo（2 - 3占有率），次要位点的褶皱为G1′-endo（1 - 3占有率）。主要位点的方向通过3′-末端鸟嘌呤和His 92（催化所必需的氨基酸）之间的堆积相互作用而稳定。这可能解释了观察到的2′，5′-GpG的高抑制率，其超过了所有其他2′，5′-GpN型抑制剂。根据距离判据，在电子密度图中有一个溶剂峰被确定为金属离子，可能是Ca 2+。该离子由两个Asp 15羧酸氧原子和六个水分子配位。配位多面体显示近似4 m2对称性。
The enzyme ribonuclease T 1 (RNase T 1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2′, 5′-guanosine (2′, 5′-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3σ in the resolution range 6 to 1.8 Å. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T 1/2′, 5′-GpG complex and of the near-isomorphous RNase T 1/2′-GMP complex are comparable. The root-mean-square deviation is 0.73 Å between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5′-terminal guanine of 2′, 5′-GpG binds to RNase t 1 in much the same way as in the 2′-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3′-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2′, 5′-GpG (except the 5′-terminal guanine), which originates from differences in the pucker of the 5′-terminal ribose. The pucker is C2′-exo for the major site (2 3 occupancy) and G1′-endo for the minor site (1 3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3′-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2′, 5′-GpG, which exceeds that of all other inhibitors of type 2′, 5′-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca 2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.