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

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

“…The maintenance of the hexameric form there, despite the lack of the entire dimer ␤-strand interface, is somehow compatible with the association of DOI into a R 6 hexamer observed in the presence of Zn 2ϩ and cyclohexanol (29). Although the T 6 3 T 3 R 3 3 R 6 dynamic transitions are rather well described by the SMB model (51)(52)(53)(54)(55)(56), the conformational events on the monomer 3 dimer 3 hexamer pathway are much less understood. Our results presented here provide further evidence that the nature of the changes on the insulin dimer interfaces (and associated other parts of the insulin molecule) is quite asymmetrical (Fig.…”

1H{19F} NOE NMR Structural Signatures of the Insulin R6Hexamer: Evidence of a Capped HisB10 Site in Aryl‐ and Arylacryloyl‐carboxylate Complexes

“…[3,4] The stability and dynamic properties of human insulin (HI) zinc hexamer formulations are critically influenced by allosteric effectors. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Therefore, the discovery of ligands with enhanced allosteric and/or pharmacological properties is important for the design of improved formulations. [3,4] Insulin hexamers exhibit positive and negative cooperativity and half-of-the-sites reactivity in ligand binding.…”

“…[3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] The HI hexamer undergoes allosteric transitions among three wellcharacterized protein conformations, which are designated T 6 , T 3 R 3 , and R 6 . [5][6][7][8][9][10][11][12][13][14][15][16][17] Crystalline and precipitated T 3 R 3 and R 6 hexamers are formulated as slow-release forms, [3,4] and are stabilized by the binding of allosteric ligands at two loci, the "phenolic pockets" (3 in T 3 R 3 and 6 in R 6 ), and the "HisB10 zinc sites" (1 in T 3 R 3 and, 2 in R 6 ). [3,4,[13][14][15][16] The Rstate HisB10 sites ( Figure 1) bind monovalent anions (halides, pseudo halides, and carboxylates).…”

“…[5][6][7][8][9][10][11][12][13][14][15][16][17] Crystalline and precipitated T 3 R 3 and R 6 hexamers are formulated as slow-release forms, [3,4] and are stabilized by the binding of allosteric ligands at two loci, the "phenolic pockets" (3 in T 3 R 3 and 6 in R 6 ), and the "HisB10 zinc sites" (1 in T 3 R 3 and, 2 in R 6 ). [3,4,[13][14][15][16] The Rstate HisB10 sites ( Figure 1) bind monovalent anions (halides, pseudo halides, and carboxylates). [3,4,[13][14][15][16] Binding interactions are structurally well characterized at the phenolic pockets [9][10][11][12] but not at the HisB10 sites.…”

“…[3,4,[13][14][15][16] The Rstate HisB10 sites ( Figure 1) bind monovalent anions (halides, pseudo halides, and carboxylates). [3,4,[13][14][15][16] Binding interactions are structurally well characterized at the phenolic pockets [9][10][11][12] but not at the HisB10 sites. Each HisB10 site is formed by a threehelix bundle consisting of Bchain residues 1-9 situated around the hexamer three-fold symmetry axis, to create 12 deep amphipathic cavities.…”