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 kex ≈ 300 s(-1) (kAB ≈ 240 s(-1)/kBA ≈ 60 s(-1)) and pA/pB ≈ 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 kex ≈ 500 s(-1) (kAB ≈ 15 s(-1)/kBA ≈ 485 s(-1)) and pA/pB ≈ 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.

Fig. 1.

ERK2 phosphorylation induces chemical shift changes at the hinge region. (A) Two-dimensional (¹³C,¹H) HMQC spectra of [methyl-¹³C]ILV-labeled 0P-ERK2 (blue) and 2P-ERK2 (red) recorded at 800 MHz and 25 °C. (B) X-ray structure of 0P-ERK2 (Protein Data Bank ID code 1ERK) showing spheres representing positions of ILV methyls, with ¹³C chemical shift differences (|Δδ¹³C|) between 0P-ERK2 and 2P-ERK2 indicated by the color scale (also see Fig. S2). Unassigned ILV methyls are represented by white spheres. DEJL is the docking site motif for ERK, JNK, and LXL. (C) Structural alignment of 0P-ERK2 (blue) and 2P-ERK2 (red). The expansion shows that the side chains of L154 and I82, which have two of the largest |Δδ¹³C| (B), form hydrophobic interactions with each other and with M106 at the hinge. Structure images were prepared using PyMOL (www.pymol.org).

Fig. 2.

CPMG dispersion experiments show changes in methyl dynamics following ERK2 phosphorylation. The dispersion curves measured at 25 °C and 900 (green), 800 (blue), and 600 (red) MHz are shown for representative residues in 0P-ERK2 (Left) and 2P-ERK2 (Right). The R_ex is shown for I72 in 2P-ERK2 at 800 MHz. Lines show individual fits to the Carver–Richards equation, and the error bars were estimated from peak intensities of duplicate measurements.

Fig. 3.

ERK2 phosphorylation induces global conformational exchange in the kinase core. (A) Assigned methyls in 0P-ERK2 are shown as spheres, with k_ex values indicated by the color scale for individual fits of methyls that have R_ex > 4 s⁻¹. The asterisks and double asterisks indicate methyls having errors in k_ex > 50% and k_ex > 100%, respectively, with the latter methyls shown as black spheres. Methyls with no observable dynamics (R_ex ≈ 0 s⁻¹) are shown as white spheres. Thr183 and Tyr185 are shown as black sticks. (B) Residues in 2P-ERK2 with observable dynamics each fitted individually, colored as in A. (C) In 2P-ERK2, 19 methyls could be fit globally to a single rate constant (k_ex = 300 ± 10 s⁻¹) and population (p_A/p_B = 80/20 ± 0.6%), demonstrating global domain motion for residues throughout the kinase core. Residues in the MAP kinase insert subdomain could not be fit by a global process.

Fig. 4.

Methyl peaks reveal a slow conformational exchange process in 2P-ERK2. (A) Two-dimensional (¹³C,¹H) methyl HMQC spectra of 2P-ERK2 from 5 to 25 °C show that many methyls have two distinct peaks that are undergoing slow conformational exchange on the NMR chemical shift timescale. The black lines on the 5-°C spectra highlight the two exchanging peaks, indicating two well-populated states. (B) Overlay of spectra from 0P-ERK2 (black) and 2P-ERK2 (red) at 25 °C shows that the higher-populated state in 0P-ERK2 corresponds to the lower-populated state in 2P-ERK2.

Fig. 5.

ME/GG hinge mutations induce conformational exchange in the kinase core but not the MAP kinase insert. (A) Overlays of several methyl regions of the 2D HMQC spectra showing similar chemical shifts for these methyls in 0P-ERK2 (blue) and ME/GG-ERK2 (red). (B) The |Δδ¹³C| between 0P-ERK2 and ME/GG-ERK2 are indicated by the color scale, and unassigned ILV methyls are represented by white spheres. The |Δδ¹³C| between 0P-ERK2 and ME/GG-ERK2 are small throughout the protein, except for residues close to the mutated M106 and E107 (shown in pink). (C) Sixteen methyls in ME/GG-ERK2 could be globally fit with k_ex (500 ± 60 s⁻¹; shown as cyan spheres) and population (97 and 3 ± 0.2%), indicating a single exchange process throughout the kinase core, similar to 2P-ERK2. The assigned methyls in the MAP kinase insert are shown as spheres, with k_ex values indicated by the color scale.

Fig. 6.

Model for activation of ERK2, involving the release of constraints to domain movement and change in energy landscape induced by phosphorylation. (A and B) Before phosphorylation, 0P-ERK2 is in an inactive conformation that is constrained from domain motion involving rigidity at the hinge (illustrated by the straight thick line between hinge residues M and E). The CPMG dispersion data show no evidence for additional conformations, indicating they must have low populations (<0.5%), reflecting ΔG° > +3.1 kcal/mol. Phosphorylation enhances hinge mobility (illustrated by the wavy thin line) and shifts the equilibrium to favor the active conformer with ΔG° = −0.8 kcal/mol and rate constants of k_AB = 240 s⁻¹ and k_BA = 60 s⁻¹. The ME/GG mutation partially relieves the constraint to domain motion by increasing mobility at the hinge, sampling of the active-like conformer (3%), and reducing ΔG° to +2.1 kcal/mol. The activation energy is not known for 0P-ERK2, indicated by the dashed line in B. (C) 0P-ERK2 and 2P-ERK2 structures show how phosphorylation at the activation loop promotes interactions between the N- and C-terminal domains through ion pairing between pT183 and Arg65/68 in helix αC, which may stabilize the active form. The ERK2 backbone is colored white, phosphates are colored orange, Thr183 and Tyr185 are colored red, Arg side chains are represented as sticks, and surfaces are shown for the guanidinium groups.