NMR dynamics investigation of ligand-induced changes of main and side-chain arginine N-H's in human phosphomevalonate kinase.
NMR dynamics investigation of ligand-induced changes of main and side-chain arginine N-H's in human phosphomevalonate kinase.
复制标题
人磷酸甲羟戊酸激酶中配体诱导的主链和侧链精氨酸 N-H 变化的 NMR 动力学研究。
DOI：
10.1021/ja906244j
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发表时间：
2010
影响因子：
15
通讯作者：
Sem,DanielS
中科院分区：
文献类型：
--
作者：
Olson,AndrewL;Cai,Sheng;Herdendorf,TimothyJ;Miziorko,HenryM;Sem,DanielS
磷酸甲羟戊酸激酶（PMK）在合成胆固醇和其它类异戊二烯的途径上催化磷酸基从三磷酸腺苷（ATP）转移到5-磷酸甲羟戊酸（M5 P）。为了允许这种反应，它的底物必须靠近，这将导致负电荷的显著和排斥性的积累。为了完成这项艰巨的任务，PMK含有17个赖氨酸和8个赖氨酸。然而，从结构和动力学的角度来看，实现这种电荷中和和结合的方式是未知的。更广泛地说，精氨酸侧链动力学在带电底物结合中的作用迄今为止尚未在实验上对任何蛋白质进行定义。在这里，我们报告了一个表征的变化的动态状态的精氨酸侧链PMK由于其高度带电的底物，ATP和M5 P的结合。这些研究通过使用甘氨酸选择性标记以消除光谱重叠来促进。无模型分析表明，虽然底物结合对精氨酸主链动力学几乎没有影响，但任一底物的结合导致整个蛋白质中精氨酸侧链的显著硬化，即使是距离结合位点>8 μ m的那些。精氨酸侧链的这种整体硬化是前所未有的，这表明存在足够强度的长程静电相互作用来限制精氨酸侧链在皮秒至纳秒时间尺度上的运动。这将是有趣的，看看这样的效果是否是一般的精氨酸残基的蛋白质，结合高度带电的基板，一旦精氨酸侧链动力学的额外的研究报告。
Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from adenosine triphosphate (ATP) to mevalonate 5-phosphate (M5P) on the pathway for synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines and eight lysines. However, the way in which this charge neutralization and binding is achieved, from a structural and dynamics perspective, is not known. More broadly, the role of arginine side-chain dynamics in binding of charged substrates has not been experimentally defined for any protein to date. Herein we report a characterization of changes to the dynamical state of the arginine side chains in PMK due to binding of its highly charged substrates, ATP and M5P. These studies were facilitated by the use of arginine-selective labeling to eliminate spectral overlap. Model-free analysis indicated that while substrate binding has little effect on the arginine backbone dynamics, binding of either substrate leads to significant rigidification of the arginine side chains throughout the protein, even those that are >8 Å from the binding site. Such a global rigidification of arginine side chains is unprecedented and suggests that there are long-range electrostatic interactions of sufficient strength to restrict the motion of arginine side chains on the picosecond-to-nanosecond time scale. It will be interesting to see whether such effects are general for arginine residues in proteins that bind highly charged substrates, once additional studies of arginine side-chain dynamics are reported.