Koepke J, Maslowska M, Heinemann U, Saenger W
Institut für Kristallographie, Freie Universität Berlin, F.R.G.
J Mol Biol. 1989 Apr 5;206(3):475-88. doi: 10.1016/0022-2836(89)90495-6.
The enzyme ribonuclease T1 (RNase T1) 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 3 sigma in the resolution range 6 to 1.8 A. 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 T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A 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 T1 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 C1'-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 Ca2+. 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.
从米曲霉中分离出的核糖核酸酶T1（RNase T1）与特异性抑制剂鸟苷酰-2',5'-鸟苷（2',5'-GpG）进行了共结晶，并通过立体化学约束最小二乘法精修结构，对于分辨率范围在6至1.8埃、X射线数据大于3σ的情况，晶体学R因子为14.9%。精修后的模型包含781个蛋白质原子、主位点的43个抑制剂原子和次位点的29个抑制剂原子、107个水分子氧原子以及一个指定为Ca的金属位点。在精修结束时，基于二维核磁共振数据对His、Asn和Gln侧链的取向进行了重新解释。RNase T1/2',5'-GpG复合物以及近同晶型的RNase T1/2'-GMP复合物的晶体堆积和酶构象具有可比性。等效蛋白质原子之间的均方根偏差为0.73埃。晶胞尺寸的差异主要归因于结合的抑制剂。2',5'-GpG的5'-末端鸟嘌呤与RNase T1的结合方式与2'-GMP复合物中的大致相同。相比之下，催化中心与磷酸基团之间的氢键不同，且3'-末端鸟嘌呤与酶不形成氢键。这种较弱的结合反映在2',5'-GpG（5'-末端鸟嘌呤除外）的2倍无序上，这源于5'-末端核糖的皱折差异。主位点的皱折为C2'-外向型（占据2/3），次位点的皱折为C1'-内向型（占据1/3）。主位点的取向通过3'-末端鸟嘌呤与His92之间的堆积相互作用得以稳定，His92是催化所需的氨基酸。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类型的抑制剂。基于距离标准，电子密度中的一个溶剂峰被确定为金属离子，可能是Ca2+。该离子由两个Asp15的羧基氧原子和六个水分子配位。配位多面体呈现近似4m2对称性。
