Correction to Effect of Cofactor Binding and Loop Conformation on Side Chain Methyl Dynamics in Dihydrofolate Reductase
Correction to Effect of Cofactor Binding and Loop Conformation on Side Chain Methyl Dynamics in Dihydrofolate Reductase
复制标题
修正辅因子结合和环构象对二氢叶酸还原酶侧链甲基动力学的影响
DOI：
10.1021/bi4003277
复制
发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
P. Wright
中科院分区：
文献类型：
--
作者：
J. Schnell;H. Dyson;P. Wright
二氢叶酸还原酶（DHFR）具有几个促进配体结合和催化的柔性活性位点环。以往的研究表明，在几个复合物的DHFR骨架动力学的时间尺度和运动幅度取决于活性位点环的构象。在这项研究中，动力学的信息扩展到含甲基的侧链。为了了解侧链动力学在配体结合和环构象中的作用，测量了二元叶酸和三元叶酸：NADP+复合物中大肠杆菌DHFR的甲基氘弛豫速率，以及通过测量3 JC γ CO和3 JC γ N偶合常数确定的苏氨酸、异亮氨酸和缬氨酸残基的χ 1旋转异构体群体。结果表明，除了在腺苷结合位点的骨架运动限制，在活性位点和周围的活性位点环的侧链的灵活性减少后，结合NADP+。在活性位点和周围的活性位点环的几个甲基的共振严重扩大叶酸：NADP+三元复合物，表明存在的化学位移时间尺度上的运动。Ile 14和Ile 94的侧链分别与辅因子和底物的烟酰胺环和蝶呤环包装在一起，在三元叶酸：NADP+复合物中表现出旋转异构体紊乱。这些侧链的构象波动可能在过渡态稳定中发挥作用; Ile 14观察到的谱线加宽表明在微秒/毫秒时间尺度上的运动。
Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates ofEscherichia coliDHFR in binary folate and ternary folate:NADP+complexes have been measured, together with χ1rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of3JCγCOand3JCγNcoupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.