HACHTUNGTRENNUNG{F} NOE NMR Structural Signatures of the Insulin R6 Hexamer: Evidence of a Capped HisB10 Site in Aryl- and Arylacryloyl-carboxylate Complexes

Although transplantation and stem cell therapies look promising, at present, type 1 (insulin-dependent) diabetics can be treated only with insulin or insulin analogues. Hexameric insulin is widely used in pharmaceutical formulations for treatment of insulin-dependent diabetes mellitus. 4] The stability and dynamic properties of human insulin (HI) zinc hexamer formulations are critically influenced by allosteric effectors. Therefore, the discovery of ligands with enhanced allosteric and/or pharmacological properties is important for the design of improved formulations. 4] Insulin hexamers exhibit positive and negative cooperativity and half-of-the-sites reactivity in ligand binding. The HI hexamer undergoes allosteric transitions among three wellcharacterized protein conformations, which are designated T6, T3R3, and R6. [5–17] Crystalline and precipitated T3R3 and R6 hexamers are formulated as slow-release forms, and are stabilized by the binding of allosteric ligands at two loci, the “phenolic pockets” (3 in T3R3 and 6 in R6), and the “HisB10 zinc sites” (1 in T3R3 and, 2 in R6). [3,4, 13–16] The Rstate HisB10 sites (Figure 1) bind monovalent anions (halides, pseudo halides, and carboxylates). 4, 13–16] Binding interactions are structurally well characterized at the phenolic pockets 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. 9–12] These cavities extend from the protein surface to the HisB10 Zn (Figure 1). Monoanions bind in these cavities and coordinate to Zn and give pseudotetrahedral Zn–(His)3-X complexes (X =Cl or SCN ), (Figure 1) or 5-coordinate complexes with carboxylates. This H ACHTUNGTRENNUNG{F} NOE study was undertaken to identify weak bonding interactions between the ligand and the cavity residues that stabilize the binding of organic carboxylates to the HisB10 zinc sites. 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; [6,8,16,18–21] 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. 19–21] The T3R3 and R6 Cl or SCN complexes (Figure 1) give HisB10 sites with the