Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201, USA.
J Am Chem Soc. 2010 Feb 24;132(7):2102-3. doi: 10.1021/ja906244j.
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 A 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.
磷酸甲羟戊酸激酶（PMK）在合成胆固醇和其他异戊二烯的途径中催化三磷酸腺苷（ATP）向甲羟戊酸 5-磷酸（M5P）的磷酸转移。为了允许这种反应发生，其底物必须靠近，这将导致显著的、排斥的负电荷积累。为了促进这一艰巨的任务，PMK 包含 17 个精氨酸和 8 个赖氨酸。然而，从结构和动力学的角度来看，这种电荷中和和结合是如何实现的还不得而知。更广泛地说，到目前为止，还没有实验定义精氨酸侧链动力学在任何蛋白质结合带电底物中的作用。本文报道了 PMK 中精氨酸侧链动力学状态因结合其带高电荷的底物 ATP 和 M5P 而发生变化的特征。这些研究通过使用精氨酸选择性标记来消除光谱重叠来进行。无模型分析表明，尽管底物结合对精氨酸骨架动力学几乎没有影响，但结合任一底物都会导致整个蛋白质中精氨酸侧链的刚性显著增加，即使是距离结合位点 >8 A 的精氨酸侧链也是如此。这种精氨酸侧链的全局僵化是前所未有的，表明存在足够强度的长程静电相互作用，以限制精氨酸侧链在皮秒到纳秒时间尺度上的运动。一旦有更多关于精氨酸侧链动力学的研究报告，看看这种效应是否普遍存在于结合高电荷底物的蛋白质中的精氨酸残基，将会很有趣。
