Figure 5. Molecular dynamics simulations of activated mutants show increased flexibility at the glycine rich loop and loop 16, and increased salt-bridge formation at the catalytic site.

(A) Structure of ERK2 with relevant features annotated: L16 – orange, Gly loop - dark orange, C spine - dark blue, R spine - light blue, gatekeeper residue – purple, catalytic residues (K52, D147 and D165) – red, and loop insertion position – green. (B–D) Root mean square fluctuations (RMSF, error bars indicating 95% confidence intervals of the means are plotted but are too small to see): Wild type – green, mutant – blue and difference between mutant and WT – red. (B) ERK2-ins55N (ß3-αC loop insertion mutant) (C) ERK2-Q103M (gatekeeper residue mutant) (D) ERK2-ins55N-Q103M (double mutant). (E) Probability density of salt-bridge between ß3-lysine and αC-glutamate, K52–E69. (F) Distribution of domain closure angles. (G) Probability density of distances < 4 Å between Y185 of the ‘TxY’ motif and active site D147H: Wild type – green, ERK2-ins55N – blue, ERK2-Q103M – yellow and ERK2-ins55N-Q103M – black. (H). Top 100 conformations of ERK2-WT with smallest Y185-D147 distance. D147 (red) and Y185 (blue) are shown as spheres.

Figure 5—figure supplement 1. Reversion of evolutionary mutations also activates ERK2.

Kinase assays measuring activity of modern ERK2 and ERK2 with loop insertion and gatekeeper mutations that convert the kinase to inferred ancestral states. MBP was used as a substrate.

Figure 5—figure supplement 2. Distribution of trajectory lengths.

3999 trajectories were collected in total and propagated until at least 500 μs of simulation time for each system had been collected, resulting in the grand total of 2.05 ms of simulation time and 4,104,841 frames. The average trajectory length was 513 ns.

Figure 5—figure supplement 3. Probability densities of various inter-residue distances in the catalytic site.

(A, B), catalytic spine (C–E) and activation segment (F, G) in ERK2 WT and mutants. Wild type (WT) – green, ERK2-ins55N – blue, ERK2-Q103M – yellow and ERK2-ins55N + Q103M – black.

Figure 5—figure supplement 4. Differential contacts between ERK2 mutants and wild type (WT).

(A–C) Differential contact maps. Red represents gain of contact probability in mutant and blue represents decreased contact probability. (D–F) Structural map of contact changes. The sum of absolute contact changes is projected onto alpha carbons and the color scheme follows a standard rainbow 0.25–2 scale.

Figure 5—figure supplement 5. A 3-State Markov state model (MSM) indicates high contact disruption along the activation segment in the ERK2-Q103M mutant.

Each state is represented by 100 structures (smoothed) from the ERK2-Q103M system, colored by the absolute sum of contact changes per residue compared to WT, following the standard rainbow on a 0.25–2 scale. The Q103M mutation is represented by a red stick model. The frequency of each state is provided in the bar graph.

Figure 5—figure supplement 6. A 4-State Markov state model (MSM) indicates an increase in the open conformation of loop 16 in insertion mutants.

Each state is represented by 100 structures (smoothed) from the ERK2-ins55N system, colored by the absolute sum of contact changes per residue compared to WT, following the standard rainbow on 0.25–2 scale. Mutation and insertion mutations are represented by red sticks. The frequency of each state is provided in the bar graph.

Figure 5—figure supplement 7. Major contact changes with Y185 mapped on to the structure of mutants.

(A–C) Residues that lose and gain contact with the Y185 in mutants compared to WT are projected onto alpha carbons of the ERK2 structure on a −0.25 to +0.25 scale. (A) ERK2-ins55N – WT, (B) ERK2-Q103M – WT and (C) ERK2-ins55N-Q103M – WT. Y185 is represented in sticks.