Phosphorylation-dependent changes in structure and dynamics in ERK2 detected by SDSL and EPR
Phosphorylation-dependent changes in structure and dynamics in ERK2 detected by SDSL and EPR
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DOI：
10.1016/s0006-3495(04)74115-6
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发表时间：
2004-01-01
影响因子：
3.4
通讯作者：
Ahn, NG
中科院分区：
文献类型：
--
作者：
Hoofnagle, AN;Stoner, JW;Ahn, NG
丝裂原活化蛋白激酶受活性位点裂隙附近两个磷酸化位点的占据率调节。先前使用氢交换来研究经典的促分裂原活化蛋白激酶（细胞外信号调节蛋白激酶-2）的研究已经表明，磷酸化改变了磷酸化位点远端>10埃的骨架构象迁移率，包括氨基酸102-105内的迁移率降低和108-109内的迁移率增加。为了进一步描述酶活化后的变化，使用氨基酸101、105-109、111、112处的定点自旋标记和电子顺磁共振光谱来研究该区域。玻璃样品中的自旋标记的各向异性超精细分裂没有改变磷酸化，与以前的晶体学研究表明在该地区没有结构变化相一致。在位置101、111和112处，自旋标记的迁移率未被二磷酸化改变，与很少或没有构象变化一致。然而，二磷酸化引起的旋转扩散率在位置105-108和改变探针的比例在一个运动约束状态在位置105，107和109小，但显着的变化。因此，电子顺磁共振表明纳秒侧链迁移率的可重复的变化，在特定的残基内的域间区域，远离磷酸化和构象变化的网站。
Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 Angstrom distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.