Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution.
Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution.
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迈向基于结构的药物设计：甘氨酰胺核糖核苷酸转化酰酶多底物加合物复合物的晶体结构，分辨率为 1.96 A。
DOI：
10.1006/jmbi.1995.0286
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Wilson,IA
中科院分区：
文献类型：
--
作者：
Klein,C;Chen,P;Arevalo,JH;Stura,EA;Marolewski,A;Warren,MS;Benkovic,SJ;Wilson,IA
用多底物加合物BW1476U89测定了大肠杆菌甘氨酸酰胺核糖核苷酸转化酶（gal - tfase; EC 2.1.2.2.）抑制剂复合物结构，r值为19.1%，分辨率为1.96 Å。利用两个不同的单斜晶格的数据，并从浸泡在20% （w/v）甲基戊二醇作为冷冻保护剂的晶体中收集数据，在- 150°C下进行冲击冷冻，通过分子和单同构置换的结合来确定结构。多底物加合物结合在酶的两个功能域之间的界面上的扩展缝隙中。该抑制剂通过与磷酸盐、谷氨酸和嘧啶环基团的三组紧密相互作用定位于结合位点，而其中间连接原子更灵活，并采用两组不同的构象。高度保守的Arg103、His108和Gln170残基是配体结合和催化（His108）的关键，它们具有代偿性构象变化，这为它们在底物特异性和甲酰基转移中的作用提供了线索。1476U89的分子设计与天然10-甲酰四氢叶酸（10-CHO-H4F, pH 8.5时Km≈77.4 μM）和甘氨酰胺- rna核苷酸（GAR, pH 8.5时Km≈8.1 μM）底物的形状、分子相互作用和结合常数非常接近，证实了其作为多底物加合物抑制剂（Ki ~ 100 pM, pH 8.5）的分子设计。这种配体复合物的立体化学表明，His108可能是一种亲电试剂，稳定了GAR的氨基直接攻击10-CHO-H4F而形成的四面体中间体的氧阴离子。对GAR-Tfase、二氢叶酸还原酶和胸苷酸合成酶的叶酸结合模式进行结构比较，发现与GAR-Tfase结合的叶酸衍生物采用不同的C-6和C-9原子间二面角的转化构象，为靶向特定的叶酸依赖酶提供了一个柄。从结合位点的两种不同的配体离散构象中获得的结构信息也为GAR-Tfase抑制剂的新设计提供了一些线索，这些抑制剂可能发展成为有用的化疗药物。
An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2.) fromEscherichia colihas been determined with a multisubstrate adduct BW1476U89 to anR-value of 19.1% at 1.96 Å resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at −150 °C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its intervening linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubtrate adduct inhibitor (Ki∼100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate(10-CHO-H4F;Km≈77.4 μM at pH 8.5) and glycinamide-ribonucleotide(GAR;Km≈8.1 μM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivates bound to GAR-Tfase differentially adopt thetransconformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for thede novodesign of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.