Autophosphorylation dependent destabilization of the insulin receptor kinase domain: Tryptophan-1175 reports changes in the catalytic cleft

Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R-953- S-1355) were excited at a lambda(ex) of 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the lambda(em) range of 310-420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss-of similar to 2.3 kcal/mol of stabilization for the first transition and similar to 1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W-1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W-1175 is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W-1175 was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W-1175 exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.