Phosphorylation releases constraints to domain motion in ERK2

Abstract

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13 C 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.T he 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 phosphoThr 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 hydrogenexchange 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 phosph...

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Synchronous Opening and Closing Motions Are Essential for cAMP-Dependent Protein Kinase A Signaling

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Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

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