Thermodynamics of nucleotide and non-ATP-competitive inhibitor binding to MEK1 by circular dichroism and isothermal titration calorimetry.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. A wealth of information about the enzymatic activity of MEK1, its domain functions, and inhibitor action is available; however, the thermodynamic properties of the interaction between MEK1 and ligands, such as nucleotides and non-ATP-competitive inhibitors, have not been reported. This study describes the thermodynamic parameters for the binding interactions of MEK1, nucleotides, and non-ATP-competitive inhibitor complexes using temperature-dependent circular dichroism (TdCD) and isothermal titration calorimetry (ITC). Non-phosphorylated MEK1 (npMEK1) has a high affinity for both AMP-PNP and ADP (Kd approximately 2microM). The binding is enthalpically favored and Mg-dependent. The active, phosphorylated form of MEK1 (pMEK1) bound nucleotides with a similar high affinity (Kd approximately 2muM) and had a thermodynamic profile and Mg-dependence similar to that of the non-phosphorylated form. The non-ATP-competitive MEK1 inhibitors, U0126 and PD0325901, showed no preference for npMEK1 and pMEK1 by TdCD. TdCD results also showed that these inhibitors are more potent in the presence of the nucleotide than in its absence. The ternary complex, MEK1.PD0325901.nucleotide, showed synergistic binding as evidenced by a large, non-additive shift in the midpoint of the protein unfolding transition (Tm). This was apparent for both npMEK1 and pMEK1 using either ADP or AMP-PNP. ITC binding studies confirmed the synergistic binding effect. The ITC-determined affinity of nucleotide (AMP-PNP, ADP) binding to the npMEK1.PD0325901 complex was enhanced nearly 5-fold compared to nucleotide binding to npMEK1 alone. In addition, the affinity of PD0325901 binding to npMEK1.nucleotide complexes was increased nearly 10-fold relative to the affinity of PD0325901 for npMEK1 alone. These are the first thermodynamic binding studies that characterize the affinity of the allosteric non-ATP-competitive inhibitors U0126 and PD0325901 with and without the nucleotide. The results indicate these allosteric inhibitors have a dynamic range in the type of MEK1 activation states and nucleotide complexes that they can bind.