Carboxylate Ions Are Strong Allosteric Ligands for the HisB10 Sites of the R-State Insulin Hexamer

Abstract

The insulin hexamer is an allosteric protein which displays positive and negative cooperativity and half-site reactivity that is modulated by strong homotropic and heterotropic ligand binding interactions at two different loci. These loci consist of phenolic pockets situated on the dimer-dimer interfaces of T-R and R-R subunit pairs and of anion sites comprising the HisB10 metal ion sites of the R3 units of the T3R3 and R6 states. In this study, we show that suitably tailored organic carboxylates are strong allosteric effectors with relatively high affinities for the R-state HisB10 metal sites. Methods of quantifying the relative affinities of ligands for these sites in both Co(II)- and Zn(II)-substituted insulin hexamers are presented. These analyses show that, in addition to the electron density on the ion, the carboxylate affinity is influenced by polar, nonpolar, and hydrophobic interactions between substituents on the carboxylate and the amphipathic protein surface of the narrow tunnel which controls ligand access to the metal ion. Since the binding of anions to the HisB10 site makes a critically important contribution to the stability of the T3R3 and R6 forms of the insulin hexamer, the design of high-affinity ligands with a carboxylate donor for coordination to the metal ion provides an opportunity for constructing insulin formulations with improved pharmaceutical properties.

Analysis of Insulin Allostery in Solution and Solid State With FTIR

“…The coordination changes from octahedral to tetrahedral coordination geometry. 12,15,[20][21][22][23][24] The tetrahedral coordination facilitates the coordination of monovalent anions instead of water molecules. The binding of a monovalent anion to the divalent cation shows strong heterotropic interaction in the R state between the binding of anions and phenolic ligands to the hydrophobic binding pockets.…”

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

“…, [9][10][11][12][13][14][15][16][18][19][20][21] (Figure 1) or 5-coordinate complexes with carboxylates. [12][13][14][19][20][21] This…”

“…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.…”

“…[21] Because the binding locus is not in question, [8,15,16,[19][20][21] a more rigorous assignment method was not needed. Control experiments demonstrated that spin diffusion was not responsible for any of the observed NOEs (see Figures S1-S3 in the Supporting Information).…”