Activation loop plasticity and active site coupling in the MAP kinase, ERK2

Changes in the dynamics of the protein kinase, ERK2, have been shown to accompany its activation by dual phosphorylation. However, our knowledge about the conformational changes represented by these motions is incomplete. Previous NMR relaxation dispersion studies showed that active, dual-phosphorylated ERK2 undergoes global exchange between at least two energetically similar conformations. These findings, combined with measurements by hydrogen exchange mass spectrometry (HX-MS), suggested that the global conformational exchange involves motions of the activation loop (A-loop) that are coupled to regions surrounding the kinase active site. In order to better understand the contribution of dynamics to the activation of ERK2, we applied long conventional molecular dynamics (MD) simulations starting from crystal structures of active, phosphorylated (2P), and inactive, unphosphorylated (0P) ERK2. Individual trajectories were run for (5 to 25) {micro}s and totaled 727 {micro}s. The results showed that the A-loop is unexpectedly flexible in both 2P- and 0P-ERK2, and able to adopt multiple long-lived (>5 {micro}s) conformational states. Simulations starting from the X-ray structure of 2P-ERK2 (2ERK) revealed A-loop states corresponding to restrained dynamics within the N-lobe, including regions surrounding catalytic residues. One A-loop conformer forms lasting interactions with the C-terminal L16 segment and shows reduced RMSF and greater compaction in the active site. By contrast, simulations starting from the most common X-ray conformation of 0P-ERK2 (5UMO) reveal frequent excursions of A-loop residues away from a C-lobe docking site pocket and towards a new state that shows greater dynamics in the N-lobe and disorganization around the active site. Thus, the A-loop in ERK2 appears to switch between distinct conformational states that reflect allosteric coupling with the active site, likely occurring via the L16 segment. Analyses of crystal packing interactions across many structural datasets suggest that the A-loop observed in X-ray structures of ERK2 may be driven by lattice contacts and less representative of the solution structure. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.