Understanding the activation and regulation of the mitogen-activated protein kinase p38 using solution NMR spectroscopy
Enzymes, including kinases, require conformational and dynamic changes to perform their biological function. Conformational changes associated with the function are well-documented using X-ray crystallography and NMR spectroscopy. However, dynamic changes associated with allostery and catalysis are getting established. NMR spectroscopy is the method of choice for these studies as it reports directly on protein dynamics/motions across different time scales (ps to s; most of the catalytic activity occurs at this timescale).
Mitogen-activated protein kinases (MAPKs), including p38, belong to the family of ser/thr protein kinases that are essential for cell differentiation and autophagy. p38 becomes activated by phosphorylation of two residues (TxY) in its activation loop. Substrate binding occurs at a recognition sequence, commonly referred to as the D-motif or the Kinase Interaction Motif (KIM). Despite extensive efforts, the molecular mechanism of activation, including how p38 dynamics are correlated with activity and regulation, remains unclear. Here, the results of auto-correlated 15N fast (ns-ps) and intermediate timescale (s-ms) dynamics measurements to understand the activation and regulation of p38 will be presented. NMR relaxation measurements were performed on the different states along the activation pathway of p38, which includes phosphorylation, substrate, and ATP binding. Unexpectedly, our data showed that phosphorylation of p38 is important, but substrate binding is indeed key for the synchronization of the dynamics across the molecule to activate p38. And, it is this synchronization that allows for enhanced ATP recruitment and thus, the full activity of p38. Besides, the regulation of MAPKs is achieved via a plethora of regulatory proteins, including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins that include MAPK phosphatases, use identical interaction sites on MAPKs, they use distinct kinase interaction motif (KIM) sequences that are present in linear, peptide-like, or well-folded protein domains. To understand how the MAPK phosphatases, interact with substrates and elucidate the structural basis for the specificity of these regulatory proteins, we studied the interaction of the MAPK binding domain of a dual-specificity phosphatase (DUSP16) with p38, using integrated structural biology approaches the results of which, will also be presented.