Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38α MAP kinase

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38 alpha 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 p38 alpha 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 p38 alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38 alpha and X-ray structures of phosphorylated p38 gamma. Conformational differences account for altered FIX 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 (alpha F-alpha G, beta 7-beta 8), the C-terminal core (alpha E), and the N-terminal core region (beta 4-beta 5, alpha L16, alpha C). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38 alpha. 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 p38 alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38 alpha 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 p38 alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence andtertiary structure, they have distinct mechanisms for dynamic control of enzyme function. (C) 2008 Elsevier Ltd. All rights reserved.