Hakoshima T, Itoh T, Tomita K, Goda K, Nishikawa S, Morioka H, Uesugi S, Ohtsuka E, Ikehara M
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
J Mol Biol. 1992 Feb 20;223(4):1013-28. doi: 10.1016/0022-2836(92)90259-m.
The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.
已确定与不可识别的核糖核苷酸2'-AMP复合的突变核糖核酸酶T1（Y45W）的晶体结构，并使用1.7埃分辨率的X射线衍射数据将其精修至R因子为0.159。还确定了该酶与2'-GMP的特定复合物，并在1.9埃分辨率下将其精修至R因子为0.173。发现2'-AMP的腺嘌呤碱基位于一个与2'-GMP的鸟嘌呤碱基结合的鸟嘌呤识别位点相距很远的碱基结合位点。腺嘌呤碱基的结合由单个氢键以及该碱基与His92的咪唑环的堆积相互作用介导。腺嘌呤碱基与His92的堆积模式类似于Koepke等人报道的核糖核酸酶T1与鸟苷酰-2',5'-鸟苷复合物中观察到的鸟嘌呤碱基的堆积，Lenz等人报道的两个鸟嘌呤碱基的堆积，以及Baudet和Janin报道的barnase与d（GpC）复合物中的堆积。这些观察结果表明该位点对碱基结合是非特异性的。2'-AMP的磷酸基团以与2'-GMP类似的方式通过与酶的七个氢键紧密锁定在催化位点。此外，2'-AMP的糖部分与酶之间形成了两个氢键。2'-AMP分子采用糖苷键的反式构象和C-3'-外向糖折叠，而2'-GMP处于具有C-3'-内向-C'-2'-外向折叠的顺式构象。该突变通过糖环的构象变化和磷酸基团从野生型酶复合物中的相应位置向催化位点内部的位移增强了2'-GMP的结合。对获得的两种晶体结构的比较解决了与核酸分解活性中的高特异性相比，不可识别的核苷酸对该酶表现出意外强结合的问题。结果表明，在鸟嘌呤识别位点将鸟嘌呤碱基与其他核苷酸碱基区分开来比迄今为止从核苷酸结合实验估计的更有效。
