The binding constant, Kb, and the binding enthalpy (DbHo) of the inhibitor drugs, SB 203580, SKF 86002, and p38 INH.1 binding to two splice variants of p38a MAP kinase and their C162S mutants in 5mM HEPES buffer at pH =7.4 were determined from isothermal titration calorimetry measurements from 25 oC to 35 oC. The splice variants are the result of one of two adjacent exons of the same length spliced out of either the main isoform, p38a isoform 2, or the other isoform, p38a isoform 1. The binding reactions are all totally enthalpically-driven with the binding affinities to in the order of p38 INH.1 >> SB 203580 > SKF 86002 and ranging from 1.30 X 1014 M-1 to 1.2+0.2 X 107 M-1 with the corresponding binding enthalpies ranging from -84+3 kJ mol-1 to -54+3 kJ mol-1 at 25 oC. Differences in the binding affinities between the 2 isoforms and their mutants become more apparent with the higher affinity binding drug inhibitors, p38 INH.1 and SB 203580 and at 30 oC and 35 oC. Differential scanning calorimetry (DSC) scans exhibited single unfolding transitions for each all four proteins, for p38a isoform 2 complexed with SKF 86002, and for the C162S mutants bound to the drug inhibitors. However, two transitions were observed for p38a isoform 1 bound to SB 203580 and for p38a isoform 2 bound to SB 203580 and to p38 INH.1. The thermodynamic differences in the binding affinities and the conformational stabilities of the p38a isoforms can be described by an equilibrium between an open conformation where there is less interaction between the amino and carboxyl terminal domains of the protein and a closed conformation favored by the C162S mutation.
Citation: Journal of Biological Chemistry
Pub Type: Journals
differential scanning calorimetry, drugs, enzymes, inhibitors, isothermal titration calorimetry, thermodynamics