School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea.
Protein Pept Lett. 2021;28(5):481-488. doi: 10.2174/0929866527666201103152534.
Activation of mitogen-activated protein kinases (MAPKs) is regulated by a phosphorylation cascade comprising three kinases, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP2K1 and MAPK2K2, also known as MEK1 and MEK2, activate ERK1 and ERK2. The structure of the MAPK signaling cascade has been studied, but high-resolution structural studies of MAP2Ks have often focused on kinase domains or docking sites, but not on full-length proteins.
To understand the conformational dynamics of MEK1.
Full-length MEK1 was purified from Escherichia coli (BL21), and its conformational dynamics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The effects of ATP binding were examined by co-incubating MEK1 and adenylyl-imidodiphosphate (AMP- PNP), a non-hydrolysable ATP analog.
MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through β1, αI, and the C-terminal helix. AMP-PNP binding was found to reduce conformational dynamics within the glycine-rich loop and regions near the DFG motif, along with the activation lip.
We report for the first time that MEK1 has regions that slowly change its folded and unfolded states (mixed EX1/EX2 kinetics) and also report the conformational effects of ATP-binding to MEK1.
丝裂原活化蛋白激酶（MAPKs）的激活受磷酸化级联反应调控，该级联反应包含三种激酶：丝裂原活化蛋白激酶激酶激酶（MAP3K）、丝裂原活化蛋白激酶激酶（MAP2K）和丝裂原活化蛋白激酶（MAPK）。MAP2K1 和 MAPK2K2，也称为 MEK1 和 MEK2，可激活 ERK1 和 ERK2。MAPK 信号转导级联的结构已得到研究，但 MAP2Ks 的高分辨率结构研究通常集中在激酶结构域或对接位点上，而不是全长蛋白上。
了解 MEK1 的构象动力学。
从大肠杆菌（BL21）中纯化全长 MEK1，并使用氢/氘交换质谱（HDX-MS）分析其构象动力学。通过共孵育 MEK1 和非水解型 ATP 类似物腺苷酰亚胺二磷酸（AMP-PNP）来检查 ATP 结合的影响。
MEK1 在 N 端尾部的β1、αI 和 C 端螺旋内表现出混合 EX1/EX2 的 HDX 动力学。发现 AMP-PNP 结合可降低甘氨酸丰富环和靠近 DFG 基序区域以及激活环的构象动力学。
我们首次报道 MEK1 具有缓慢改变其折叠和未折叠状态的区域（混合 EX1/EX2 动力学），并报告了 ATP 结合对 MEK1 的构象影响。
