STRUCTURE, MULTIPLE SITE BINDING, AND SEGMENTAL ACCOMMODATION IN THYMIDYLATE SYNTHASE ON BINDING DUMP AND AN ANTIFOLATE
STRUCTURE, MULTIPLE SITE BINDING, AND SEGMENTAL ACCOMMODATION IN THYMIDYLATE SYNTHASE ON BINDING DUMP AND AN ANTIFOLATE
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DOI：
10.1021/bi00482a004
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发表时间：
1990-07-31
期刊：
影响因子：
2.9
通讯作者：
STROUD, RM
中科院分区：
文献类型：
--
作者：
MONTFORT, WR;PERRY, KM;STROUD, RM
与底物 dUMP 和辅因子亚甲基四氢叶酸类似物复合的大肠杆菌胸苷酸合酶 (TS) 的结构通过多重同晶置换得到解析，并在 1.97-ANG 处精修。对于高度受限的结构，所有数据的残差分辨率为 18%（数据>2.sigma 为 16%）。结构中的所有残基均得到清晰解析，并且对结构的完全正确性给出了非常高的信心。三元复合物直接表明 dUMP 甲基化是如何发生的。 dUMP 的 C-6 在催化过程中与 Cys-198 (146) 的 γS 共价结合，并且反应物被特定的氢键和来自保守残基的疏水相互作用包围。与独立解析的未配体 TS 的结构相比，揭示了酶的巨大构象变化，酶关闭以将反应物和几个高度有序的水分子隔离在海绵状活性中心内，远离本体溶剂。通过在晶体储存期间停止添加还原剂，发现了辅因子类似物喹唑啉环的第二个结合位点。蛋白质中的化学变化是轻微的，并且从不同密度图来看，巯基的修饰并不直接导致初级位点的封锁。该位点仅与主要位点部分重叠，也被保守残基包围，因此可能发挥功能作用。配体诱导的构象变化不是结构域转变，而是涉及几个螺旋、β-链和环的分段调节，这些螺旋、β-链和环作为单元相对于单体之间的β-片层界面移动。
The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-.ANG. resolution to a residual of 18% for all data (16% for data >2.sigma.) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to .gamma.S of Cys-198 (146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, .beta.-strands, and loops that move as units against the .beta.-sheet interface between monomers.