Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes
Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes
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DOI：
10.1110/ps.03219603
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发表时间：
2003-10-01
期刊：
影响因子：
8
通讯作者：
Wright, PE
中科院分区：
文献类型：
--
作者：
Osborne, MJ;Venkitakrishnan, RP;Wright, PE
本文用核磁共振方法研究了四种大肠杆菌二氢叶酸还原酶（DHFR）复合物在溶液中的构象。对叶酸和氧化NADP（+）辅因子的三元复合物以及叶酸和还原辅因子类似物5，6-二氢NADPH的三元复合物进行了H-1（N）、N-15和C-13（α）共振归属。的骨干化学位移进行了比较与那些的二元复合物的DHFR与底物类似物叶酸和二元复合物与NADPH（全酶）。H-1（N）和N-15化学位移的分析导致了对酶活性位点构象的标记共振的鉴定。其他骨架酰胺共振报告在蝶呤结合口袋和NADPH辅因子的腺苷和烟酰胺-核糖结合位点中存在配体。化学位移数据表明，该酶在溶液中占据两个主要的结构状态，与活性位点环在封闭或闭塞构象定义的X射线晶体学;没有证据表明，在一些X射线结构中观察到的开放构象的E。coliDHFR在溶液中增殖。
Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. H-1(N), N-15, and C-13(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of H-1(N) and N-15 chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.