Kinase Activation by Small Conformational Changes

Protein kinases (PKs) are allosteric enzymes that play an essential role in signal transduction by regulating a variety of key cellular processes. Most PKs suffer conformational rearrangements upon phosphorylation that strongly enhance the catalytic activity. Generally, it involves the movement of the phosphorylated loop toward the active site and the rotation of the whole C-terminal lobe. However, not all kinases undergo such a large configurational change: The MAPK extracellular signal-regulated protein kinases ERK1 and ERK2 achieve a 50 000 fold increase in kinase activity with only a small motion of the C-terminal region. In the present work, we used a combination of molecular simulation tools to characterize the conformational landscape of ERK2 in the active (phosphorylated) and inactive (unphosphorylated) states in solution in agreement with NMR experiments. We show that the chemical reaction barrier is strongly dependent on ATP conformation and that the “active” low-barrier configuration is subtly regulated by phosphorylation, which stabilizes a key salt bridge between the conserved Lys52 and Glu69 belonging to helix-C and promotes binding of a second Mg ion. Our study highlights that the on–off switch embedded in the kinase fold can be regulated by small, medium, and large conformational changes.蛋白激酶（Protein kinases, PKs）是一类别构酶（allosteric enzymes），通过调控多种关键细胞进程在信号转导（signal transduction）中发挥核心作用。多数蛋白激酶在发生磷酸化（phosphorylation）后会出现构象重排（conformational rearrangements），该过程可显著增强其催化活性（catalytic activity），通常涉及磷酸化环（phosphorylated loop）向活性位点（active site）的移动，以及整个C端结构域（C-terminal lobe）的旋转。然而，并非所有激酶都会发生如此大幅度的构象变化：丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）家族的细胞外调节蛋白激酶ERK1与ERK2，仅通过C端区域的小幅运动，就能使激酶活性提升50000倍。本研究结合多种分子模拟工具（molecular simulation tools），结合核磁共振（NMR）实验结果，对溶液中处于活性（磷酸化）与非活性（未磷酸化）状态的ERK2的构象分布（conformational landscape）进行了表征。研究表明，化学反应能垒（chemical reaction barrier）与三磷酸腺苷（ATP）的构象密切相关，而"活性"低能垒构象（low-barrier configuration）受磷酸化的精细调控：磷酸化作用可稳定保守的C螺旋（helix-C）上赖氨酸52（Lys52）与谷氨酸69（Glu69）之间的关键盐桥（salt bridge），并促进第二个镁离子（Mg ion）的结合。本研究揭示，嵌入激酶折叠结构（kinase fold）中的"通断开关"（on-off switch），可通过小幅、中幅以及大幅构象变化实现调控。