Phosphorylation releases constraints to domain motion in ERK2

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR ¹³C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k_ex ≈ 300 s⁻¹ (k_AB ≈ 240 s⁻¹/k_BA ≈ 60 s⁻¹) and p_A/p_B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k_ex ≈ 500 s⁻¹ (k_AB ≈ 15 s⁻¹/k_BA ≈ 485 s⁻¹) and p_A/p_B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phospho-Thr and phospho-Tyr residues and basic residues in the N- and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogen-exchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7–9) led to a model where conformational mobility at the hinge linking the N- and C-terminal domains is increased by phosphorylation, therefore releasing constraints needed for activation. Such a model differs from other types of autoinhibitory mechanisms in protein kinases, which involve interactions with domains outside the kinase core (10, 11). However, how hinge flexibility regulates ERK2 is unknown.NMR relaxation dispersion methods enable protein dynamics to be monitored by measuring exchange between conformational states (12). In particular, Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiments report on motions on slow (100–2,000 s⁻¹) timescales (13), which are often important for enzymatic function (13–16). In the CPMG experiment, exchange between different conformational states is probed with varying times between “refocusing” pulses. Conformational exchange leads to imperfect refocusing, thus decreasing the intensity of the NMR signal. Increasing the pulse frequency allows less chance for conformational exchange, and therefore increased NMR signal intensity. For a given pulse frequency, analysis of the signal intensity yields the effective relaxation rate for the resonance, R_2,eff. This is typically plotted as a relaxation dispersion curve, which can be fit to a two-state conformational exchange process (e.g., A ⇌ B interconversion). Fitting extracts the populations and the exchange rates between states, thus reflecting the thermodynamics and kinetics of the system (17, 18).Here we performed CPMG relaxation dispersion experiments at multiple field strengths to compare the dynamic properties of [¹³C]methyl-labeled ERK2 in its phosphorylated and unphosphorylated states. The results demonstrate that phosphorylation causes a significant change in exchange dynamics throughout the kinase core, consistent with a global domain motion. Increasing hinge mobility by introducing mutations at the hinge also promotes domain motion within the core but with differing kinetics and populations. Taken together, the results show that large changes in dynamics accompany ERK2 phosphorylation, which are influenced by conformational mobility at the hinge. We propose that the activation of ERK2 involves removing inhibitory constraints to domain motion, which are conferred by the internal architecture of the kinase.