Sours Kevin M, Kwok Stan C, Rachidi Thami, Lee Thomas, Ring Adam, Hoofnagle Andrew N, Resing Katheryn A, Ahn Natalie G
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
J Mol Biol. 2008 Jun 20;379(5):1075-93. doi: 10.1016/j.jmb.2008.04.044. Epub 2008 Apr 25.
Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.
氢-氘交换测量是研究蛋白质构象和构象流动性变化的一种强大方法。在此，我们通过氢交换（HX）质谱法检测p38α丝裂原活化蛋白激酶（MAPK），以确定构象流动性的变化是否可能由激酶磷酸化和激活诱导。我们研究了影响HX质谱实验中序列覆盖率的因素，结果表明，改变采样深度、仪器和肽段搜索策略可实现可交换酰胺的最高覆盖率。p38α的区域氘化模式与三级结构一致，且与先前确定的细胞外信号调节激酶（ERK）2的氘化模式相似，这表明MAPK在局部酰胺HX程度方面具有保守性。p38α的激活改变了五个区域的HX，通过比较未磷酸化p38α的X射线结构和磷酸化p38γ的X射线结构来解释这些变化。构象差异导致激活环、P + 1位点和活性位点内的HX改变。相比之下，HX的改变归因于激活对底物对接位点（αF-αG、β7-β8）、C末端核心区域（αE）和N末端核心区域（β4-β5、αL16、αC）内构象流动性的影响。激活还降低了p38α C末端延伸处3-10螺旋中的HX。虽然ERK2中的这个螺旋形成了一个二聚化界面，在激活后会免受HX影响，但分析超速离心表明，p38α中不会发生这种情况，因为未磷酸化和双磷酸化形式均为单体。最后，单磷酸化p38α中的HX模式与未磷酸化激酶中的相似，这表明主要的激活环重塑事件仅在双磷酸化后发生。重要的是，尽管p38α和ERK2在整体氘化方面相似，但激活诱导的HX模式仍存在差异，这表明尽管MAPK在一级序列和三级结构方面密切相关，但它们在酶功能的动态控制方面具有不同的机制。
