The mitogen activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and non-canonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the sub-optimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and non-canonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and non-canonical motifs, are not restricted to the recruitment sites, but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the non-canonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB 13. This results in a 2-fold increase in k cat/K m for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k cat/K m by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k cat/K m, with little apparent change in k cat. A peptide that binds to the DRS of ERK2 affects K m, but not k cat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His6-tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~ 90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8–11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40,180 ± 240 Da (- Mg2+ ions) and 41,290 ± 330 Da (+ Mg2+ ions). These values, close to the sequence-derived mass of 41,711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~ 3.22 S for activated ERK2 with or without 10 mM MgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for a globular protein of ~ 42 kDa. The translational diffusion coefficient of ~ 8.3 × 10-7 cm2s-1 calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~ 57 kDa with the cytoplasmic scaffold protein PEA-15. Thus ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the non-physiological His6-Tag shows substantial dimerization under the same ionic conditions.

The Zn(II)/Co(II)-sensing transcriptional repressor, Staphylococcus aureus CzrA, is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N(3)O metal sensor coordination sites. A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins. Here, we exploit covalent (Gly(4)Ser)(n)() linkers of variable length in "fused" CzrAs, where n = 1 (designated 5L-fCzrA), 2 (10L-fCzrA), or 3 (15L-fCzrA), as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees. While 15L-fCzrA exhibits properties most like homodimeric CzrA, shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly. Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity. However, Zn(II) negatively regulates DNA binding of all fCzrAs, with allosteric coupling free energies (DeltaG(1)(c)) of 4.6, 3.1, and 2.7 kcal mol(-1) for 15L-, 10L-, and 5L-fCzrAs, respectively. Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in DeltaG(1)(c) = 2.6 kcal mol(-1) or approximately 83% that of 10L-fCzrA; in contrast, homodimeric H97N CzrA gives DeltaG(1)(c) = 0. (1)H-(15)N HSQC spectra acquired for wt-, 10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted. Thus, occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding.

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•LpepJIP1 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 flexible and to exhibit changes in dynamics upon L-pepJIP1 binding. The conformation of the activation loop for the apo state is similar to the inactive form of apo ERK2, while for the JNK1•L-pepJIP1 complex, it is more like the inactive form of ERK2 bound to pepHePTP. ED analysis shows that, after the binding of L-pepJIP1, the Nand 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 whose open conformation is favored, consistent with a previous study of Src and Lck kinases that interlobe opening motion was needed for the possible intramolecular self-activation. The present study suggests that L-pepJIP1 regulates the inter-domain 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 towards 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.