Structural Asymmetry and Half-Site Reactivity in the T to R Allosteric Transition of the Insulin Hexamer

Abstract

The zinc-insulin hexamer, the storage form of insulin in the pancreas, is an allosteric protein capable of undergoing transitions between three distinct conformational states, designated T6, T3R3, and R6, on the basis of their ligand binding properties, allosteric behavior, and pseudo point symmetries [Kaarsholm, N. C., Ko, H.-C., & Dunn, M. F. (1989) Biochemistry 28, 4427-4435]. The transition from the T-state to the R-state involves a coil-to-helix transition in residues 1-8 of the B-chain wherein the ring of PheB1 is displaced by approximately 30 A. This motion also is accompanied by small changes in the positions of A-chain residues and other B-chain residues. In this paper, one- and two-dimensional (COSY and NOESY) 1H NMR are used to characterize the ligand-induced T to R transitions of wild-type and EB13Q mutant human zinc-insulin hexamers and to make sequence-specific assignments of all resonances in the aromatic region of the R6 complex with resorcinol. The changes in the 1H NMR spectrum (at 500 and 600 MHz) that occur during the T to R transition provide specific signatures of the conformation change. Analysis of the dependence of these spectral changes for the phenol-induced transition as a function of the concentration of phenol establish (1) that the interconversion of T6 and R6 occurs via a third species assigned as T3R3 and (2) that the system shows both negative and positive cooperative allosteric behavior. One- and two-dimensional COSY and NOESY studies show that, in the absence of phenolic compounds, anions act as heterotropic effectors that shift the distribution of hexamer conformations in favor of the R-state with the order of effectiveness, SCN- > N3- >> I- >> Cl-. Analysis of one- and two-dimensional spectra indicate that with wild-type insulin, SCN- and N3- give T3R3 species, whereas the EB13Q mutant gives an R6 species. An allosteric model for the insulin T to R transition based on the structural asymmetry model [Seydoux, F., Malhotra, O. P., & Bernhard, S. A. (1974) CRC Crit. Rev. Biochem. 2, 227-257] is proposed that explains the negative and positive allosteric properties of the system, including the role of T3R3 and the action of homotropic and heterotropic effectors.

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

“…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. 3, B-D); thus, they fit and support well the occurrence of structural asymmetries that are important features of a SMB model for the allosteric behavior of insulin (57).…”

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.…”