Insulin Allosteric Behavior:  Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

Abstract

The insulin hexamer is an allosteric protein widely used in formulations for the treatment of diabetes. The hexamer exhibits positive and negative cooperativity and apparent half-site binding activity, reflecting the interconversion of three allosteric states, designated as T6, T3R3, and R6. The hexamer contains two symmetry-related Zn2+ located 16 A apart on the 3-fold symmetry axis. In the transition of T3 units to R3 units, Zn2+ switches from an octahedral Zn2+ N3O3 complex (N is HisB10, O is H2O) to a distorted tetrahedral Zn2+ N3L complex (L is a monovalent anion). Hence, monovalent anions are allosteric ligands that stabilize R3 units of T3R3 and R6. Herein, we exploit the high sensitivity of 19F NMR chemical shifts and fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T3R3 and R6. We show that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamate give bound resonances that distinguish between T3R3 and R6. 3-Trifluoromethylbenzoate and 2-trifluoromethylcinnamate also were shown to bind to the R3 units of T3R3 and R6 in two alternative, slowly interconverting modes with different microenvironments for the CF3 groups. Line width analysis shows that ligand off rates are slower by 1/10(3) than the diffusion limit, indicating a rate-limiting protein conformational transition. These studies confirm that the Seydoux, Malhotra, and Bernhard allosteric model (Bloom, C. R., Choi, W. E., Brzovic, P. S., Ha, J. J., Huang, S. T., Kaarsholm, N. C., and Dunn, M. F. (1995). J. Mol. Biol. 245, 324-330), provides a robust description of the insulin hexamer.

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).…”

“…The binding of carboxylates to the HisB10 site has been extensively investigated in solution, and it is unambiguously established that binding involves coordination to the HisB10 zinc ions; [6,8,16,[18][19][20][21] however, no X-ray structures of R-state carboxylate complexes have been published. Aromatic carboxylates make contacts with the HisB10 cavity walls that contribute substantially to the binding free energy.…”

“…Aromatic carboxylates make contacts with the HisB10 cavity walls that contribute substantially to the binding free energy. [3,8,[19][20][21] The T 3 R 3 and R 6 Cl À or SCN À complexes ( Figure 1) give HisB10 sites with the anion positioned on the hexamer three-fold symmetry axis and coordinated to Zn II . [9][10][11][12][22][23][24] The structure most relevant to this work, 1ZNJ, [22] has one HisB10 cavity of HI-R 6 that is occupied both by a Cl À coordinated to Zn II and by a phenol.…”

“…The image was created by using PyMOL 1.0 and PDB structure 2TCI. [10] one of three symmetry-related positions [4,8,21] throughout the crystal lattice. 19 F NMR spectroscopy of fluorinated ligands complexed to protein sites can be useful for investigating protein structurefunction relationships.…”