Understanding the specificity of a docking interaction between JNK1 and the scaffolding protein JIP1

Abstract

The up-regulation of JNK activity is associated with a number of disease states. The JNK-JIP1 interaction represents an attractive target for the inhibition of JNK-mediated signaling. In this study, molecular dynamics simulations have been performed on the apo-JNK1 and the JNK1•L-pepJIP1 and JNK1•D-pepJIP1 complexes to investigate the interaction between the JIP1 peptides and JNK1. Dynamic domain studies based on essential dynamics (ED) analysis of apo-JNK1 and the JNK1•L-pepJIP1 complex have been performed to analyze and compare details of conformational changes, hinge axes, and hinge bending regions in both structures. The activation loop, the αC helix, and the G loop are found to be highly flexible and to exhibit significant changes in dynamics upon L-pepJIP1 binding. The conformation of the activation loop for the apo state is similar to that of inactive apo-ERK2, while the activation loop in JNK1•L-pepJIP1 complex resembles that of the inactive ERK2 bound with pepHePTP. ED analysis shows that, after the binding of l-pepJIP1, the N- and C-terminal domains of JNK1 display both a closure and a twisting motion centered around the activation loop, which functions as a hinge. In contrast, no domain motion is detected for the apo state for which an open conformation is favored. The present study suggests that L-pepJIP1 regulates the interdomain motions of JNK1 and potentially the active site via an allosteric mechanism. The binding free energies of L-pepJIP1 and D-pepJIP1 to JNK1 are estimated using the molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods. The contribution of each residue at the interaction interface to the binding affinity of L-pepJIP1 with JNK1 has been analyzed by means of computational alanine-scanning mutagenesis and free energy decomposition. Several critical interactions for binding (e.g., Arg156/L-pepJIP1 and Glu329/JNK1) have been identified. The binding free energy calculation indicates that the electrostatic interaction contributes critically to specificity, rather than to binding affinity between the peptide and JNK1. Notably, the binding free energy calculations predict that D-pepJIP1 binding to JNK1 is significantly weaker than the L form, contradicting the previous suggestion that D-pepJIP1 acts as an inhibitor toward JNK1. We have performed experiments using purified JNK1 to confirm that, indeed, D-pepJIP1 does not inhibit the ability of JNK1 to phosphorylate c-Jun in vitro.

Figure 1

Cartoon diagram of the JNK1 structure highlighting the location of the L-pepJIP1 (red and sticks) (PDB ID: 1UKI) and the sequences of the scaffolding protein (L-pepJIP1). The G loop is indicated in cyan, the MAP kinase insert in yellow, αC helix in brown, the activation loop in magenta, the catalytic loop in blue, the loop connecting β5 and β6 and loop 16 in grey. The secondary structures are labeled based on reference.

Figure 2

RMSD plots for the protein backbone of the complex formed between JNK1 and pepJIP1 relative to its initial structure: (a) with activation loop; (b) without activation loop.

Figure 3

Atomic positional fluctuations of the Cα, N and C atoms of the systems.

Figure 4

Stereo view of the binding specificity determining regions for L-pepJIP1 binding with JNK1. (a) Residues identified as critical in the alanine-scanning mutagenesis are shown in stick model. (b) Surface representation of JNK1 in complex with L-pepJIP1. Selected secondary structures are labeled. The dashed lines denote hydrogen bonds.

Figure 5

Porcupine plots of the two largest PCA modes from ED analysis of JNK1. The first and second motion modes for (a, b) the JNK1•L-pepJIP1 complex and (c, d) the apo JNK1. Activation loop (blue), G loop (green), αC helix (yellow), loop 16 (ice blue), and MAP kinase insert (cyan). The arrows show mode.

Figure 6

Stereoview superposition of the activation loop (grey) of JNK1 and ERK2. (a) apo JNK1; (b) JNK1•L-pepJIP1. The inactive form of apo ERK2 (cyan), the inactive form of ERK2 in complex with pepHePTP (blue), and the active form (red) of ERK2. The activation loops are shown in cartoon. Six snapshots are extracted from the last 40 ns trajectories randomly.

Figure 7

Dynamic domain identification of the JNK1•L-pepJIP1 complex for the first (a) and second (b) principal modes from the DynDom analysis. The arrows represent the hinge axes and the direction of rotation from conformer 1 to 2 (grey). The fixed domains are shown in blue, the residues involved in inter domain bending are green, and the moving domains are red.

Figure 8

(a) Time evolution of the distance between the center of mass of the C- and N-domain of JNK1 during the production run; (b) Time evolution of the angle. (c) Definition of the distance (D) and angle (θ) for the N terminal domain with respect to the C terminal domain: The b and b’, the center of the N terminal domain of the complex and apo JNK1; The o (reference point, CA of the hinge residue Met111); the a, the center of the C terminal domain of the complex and apo JNK1; θ, the angle between oa and ob (JNK1•L-pepJIP1) or ob’ (apo).

Figure 9

IC₅₀ Curves comparing the effect of L-pepJIP1 peptide <●> (a peptide corresponding to the D-domain of JIP1 scaffold protein-amino acids 153-163) and the D-pepJIP1 peptide <■> to inhibit JNK1α1 activity in vitro toward recombinant GST-C-JUN (1-221). L-pepJIP1 showed IC₅₀ of 1 ± 0.08 μM while D-pepJIP1 did not show any inhibition even at 400 μM.

Figure 10

Ligand-residue interaction spectrum of (a) JNK1; (b) L-pepJIP1.

Figure 11

Comparison of ΔΔG_b values for L-pepJIP1 binding to JNK1 for alanine mutants. (a) JNK1; (b) L-pepJIP1. Relative binding free energy is the difference between binding free energy of wild-type JNK1 versus the alanine mutants: ΔΔG_b = ΔG_b (mut)–ΔG_b (wild), Positive numbers in ΔΔG_b mean highly unfavorable substitutions. In contrast, negative ΔΔG_b indicates the preference for alanine mutation. The units of ΔΔG_b are kcal mol^-1. All values are provided in the supporting information.