Proteomics and the Control of MAP Kinase Dynamics

Abstract

Enzyme motions control catalysis through mechanisms that are incompletely understood. Here, we use hydrogen exchange mass spectrometry (HX‐MS) and NMR relaxation to report internal motions in the MAP kinase, ERK2. HX‐MS shows that ERK2 activation by phosphorylation increases conformational mobility at the linker between N‐ and C‐terminal domains. This predicts that domain closure is regulated by phosphorylation, corroborated by showing that ERK2 binds ATP in two modes distinguishable by kinase activity state. Thus, before phosphorylation, ERK2 is constrained from interdomain interactions needed for catalysis, and this is overcome by phosphorylation, via altered linker dynamics. We next performed temperature‐dependent methyl‐Carr‐Purcell‐ Meiboom‐Gill (CPMG) NMR experiments, monitoring side chain motions by chemical exchange between conformational states in 13C‐methyl‐Ile,Leu,Val‐ERK2. CPMG‐NMR shows changes in dynamics between active and inactive ERK2 with kex in the μs‐ms time regime. The side chain motions vary widely in inactive ERK2, but in contrast, a large set of methyl groups share kinetic and thermodynamic properties in active ERK2. The active enzyme undergoes conformational exchange between two wellpopulated (80%/20%) states with kex ~300 s−1. Thus, kinase activation leads to changes in enzyme dynamics, involving concerted motions between residues surrounding the active site.