Characterization of the r-state insulin hexamer and its derivatives. The hexamer is stabilized by heterotropic ligand binding interactions

Abstract

1H NMR and UV-visible electronic absorption studies have been performed to investigate the effects of anions and cyclic organic molecules on the interconversion of the T- and R-conformational states (Kaarsholm et al., 1989) of hexameric M (II)-substituted insulin in solution (M = Zn or Co.). Two ligand binding processes that stabilize the R-state conformation of the M(II)-substituted insulin hexamer [M(II)-R6] have been distinguished: (i) The binding of neutral organic molecules to the six, crystallographically identified, protein pockets in the Zn(II)-R6 insulin hexamer (Derewenda et al. 1989) generate homotropic site-site interactions that stabilize the R-state. Cyclohexanol, phenol, 4-nitrophenol, and 4-hydroxymethylbenzoate are shown to bind at these sites. (ii) The coordination of singly charged anions that are able to gain access to the two HisB10 coordinated metal ions of the M(II)-R6 hexamer stabilizes the R-state. Adducts of the M(II)-R6 hexamer are formed, thereby, in which the solvent-accessible fourth coordination position of the M(II) ion is replaced by a competing anion. Binding to these two classes of sites introduces strong heterotropic interactions that stabilize the R-state. UV-visible spectral data and apparent affinity constants for the adducts formed by the Co(II)-R6 hexamer with a wide range of anionic ligands are presented. The Co(II)-R6 adducts have a strong preference for the formation of pseudotetrahedral Co(II) centers. The HCO3- and pyridine-2-thiolate ions form Co(II)-R6 adducts that are proposed to possess pentacoordinate Co(II) geometries. The relevance of the Co(II)-R6 complexes to carbonic anhydrase catalysis and zinc enzyme model systems is discussed.

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.…”