Insulin circulates in the bloodstream and binds to its specific cell-surface receptor as a 5808 Da monomeric species. However, studies of the monomer structure and dynamics in solution are severely limited by insulin self-association into dimers and higher oligomers. In the present work we use site-directed mutagenesis of the dimer- and hexamer-forming surfaces to yield the first insulin species amenable for structure determination at neutral pH by nuclear magnetic resonance (NMR) spectroscopy. The preferred insulin mutant, i.e., (B1, B10, B16, B27) Glu, des-B30 insulin retains 47% biological potency and remains monomeric at millimolar concentrations in aqueous solution at pH 6.5-7.5 as judged by NMR and near-UV circular dichroism (CD) spectroscopy. From a series of 2D 1H-NMR spectra collected at pH 6.5 and 34 degrees C, the majority of the resonances are assigned to specific residues in the sequence, and nuclear Overhauser enhancement (NOE) cross-peaks are identified. NOE-derived distance restraints in conjunction with torsion restraints based on measured coupling constants, 3JHNH alpha, are used for structure calculations using the hybrid method of distance geometry and simulated annealing. The calculated structures show that the major part of the insulin mutant is structurally well defined with an average root mean square (rms) deviation between the 25 calculated structures and the mean coordinates of 0.66 A for backbone atoms (A2-A19 and B4-B26) and 1.31 A for all backbone atoms. The A-chain consists of two antiparallel helices, A2-A7 and A12-A19, connected by a loop. The B-chain contains a loop region (B1-B8), an alpha-helix (B9-B19), and a type I turn (B20-B23) and terminates as an extended strand (B24-B29). The B1-B4 and B27-B29 regions are disordered in solution. The structure is generally similar to crystal structures and resembles a crystalline T-state more than an R-state in the sense that the B-chain helix is confined to residues B9-B19.

The chromophoric divalent metal ion chelators 4-(2-pyridylazo)resorcinol (PAR) and 2,2',2"-terpyridine (terpy) are used as kinetic and spectroscopic probes to investigate in solution the SCN- -induced conformational transformations of the insulin, proinsulin, and miniproinsulin hexamers (miniproinsulin is a proinsulin analogue wherein the C-chain is replaced by a dipeptide cross-link between Gly-A1 and Ala-B30). Herein we designate the 2Zn and 4Zn crystal forms of the hexamer as the T6 and T3R3 conformations, respectively. For all three proteins, addition of SCN- reduces the rate of sequestering and removal of zinc ion by chelator. The effect of SCN- on the rate of this process saturates at the same concentration (30 mM) known to induce the T6 to T3R3 transformation in the insulin crystal. Under both T6 and T3R3 conditions, the critical stoichiometry for high-affinity interaction between Zn2+ and each of the three proteins is shown to be 2 mol of Zn2+/mol of protein hexamer. Consequently, we confirm the finding that off-axial coordination of Zn2+ via His-B10 and His-B5 residues is of minor importance for the SCN- -induced conformation change in solution [Renscheidt, H., Strassburger, W., Glatter, U., Wollmer, A., Dodson, G. G., & Mercola, D. A. (1984) Eur. J. Biochem. 142, 7-14]. Under T6 conditions, the kinetics of the reactions between insulin, proinsulin, and miniproinsulin and a variable excess of terpy are similar and biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).

The allosteric behavior of the Co(II)-substituted insulin hexamer has been investigated using electronic spectroscopy to study the binding of different phenolic analogues and singly charged anions to effector sites on the protein. This work presents the first detailed, quantitative analysis of the ligand-induced T- to R-state allosteric transition of the insulin hexamer. Recent studies have established that there are two ligand binding processes which stabilize the R-state conformation of the Co(II)-substituted hexamer: the binding of cyclic organic molecules to the six protein pockets present in the Zn(II)-R6 insulin hexamer [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 594-596] and the coordination of singly charged anions to the His(B10) metal sites [Brader, M.L., Kaarsholm, N.C., Lee, W.K., & Dunn, M.F. (1991) Biochemistry 30, 6636-6645]. The R6 insulin hexamer is stabilized by heterotropic interactions between the hydrophobic protein pockets and the coordination sites of the His(B10)-bound metal ions. The binding studies with 4-hydroxybenzamide, m-cresol, resorcinol, and phenol presented herein show that, in the absence of inorganic anions, the 4-hydroxybenzamide-induced transition, with a Hill number of 2.8, is the most cooperative, followed by m-cresol, phenol, and resorcinol with Hill numbers of 1.8, 1.4, and 1.2, respectively. The relative effectiveness of these ligands in shifting the allosteric equilibrium in favor of the Co(II)-R6 hexamer was found to be resorcinol > phenol > 4-hydroxybenzamide > m-cresol.(ABSTRACT TRUNCATED AT 250 WORDS)

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