Allosteric transition of the insulin hexamer is modulated by homotropic and heterotropic interactions

The allosteric behavior of the Co(II)-substituted insulin hexamer has been investigated using electronic spectroscopy to study the binding of different phenolic analogues and singly charged anions to effector sites on the protein. This work presents the first detailed, quantitative analysis of the ligand-induced T- to R-state allosteric transition of the insulin hexamer. Recent studies have established that there are two ligand binding processes which stabilize the R-state conformation of the Co(II)-substituted hexamer: the binding of cyclic organic molecules to the six protein pockets present in the Zn(II)-R6 insulin hexamer [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 594-596] and the coordination of singly charged anions to the His(B10) metal sites [Brader, M.L., Kaarsholm, N.C., Lee, W.K., & Dunn, M.F. (1991) Biochemistry 30, 6636-6645]. The R6 insulin hexamer is stabilized by heterotropic interactions between the hydrophobic protein pockets and the coordination sites of the His(B10)-bound metal ions. The binding studies with 4-hydroxybenzamide, m-cresol, resorcinol, and phenol presented herein show that, in the absence of inorganic anions, the 4-hydroxybenzamide-induced transition, with a Hill number of 2.8, is the most cooperative, followed by m-cresol, phenol, and resorcinol with Hill numbers of 1.8, 1.4, and 1.2, respectively. The relative effectiveness of these ligands in shifting the allosteric equilibrium in favor of the Co(II)-R6 hexamer was found to be resorcinol > phenol > 4-hydroxybenzamide > m-cresol.