Half-Site Reactivity, Negative Cooperativity, and Positive Cooperativity:  Quantitative Considerations of a Plausible Model

Abstract

The nature of cooperative allosteric interactions has been the source of controversy since the ground-breaking studies of oxygen binding to hemoglobin. Until recently, quantitative examples of a model based on the inherent symmetry and asymmetry of oligomeric proteins have been lacking. This laboratory has used the phenolic ligand binding characteristics of the insulin hexamer to develop the first quantitative model for a symmetry-asymmetry-based cooperativity mechanism. The insulin hexamer possesses positive and negative heterotropic and homotropic interactions involving two classes of sites. In this study, we explore the effects of heterotropic interactions between these sites. We show that application of the pairwise structural asymmetry theory of Seydoux, Malhotra, and Bernhard (SMB) gives excellent agreement between the ligand binding behavior and X-ray crystal structure data. Furthermore, by comparing experimental data with computer simulations, we show that the insulin hexamer can be described by a three-state SMB model involving two positive homotropic cooperative transitions linked by a negative homotropic interaction. The first transition, T3T3' right harpoon over left harpoon T3oR3o, with allosteric constant LoA = [T3T3']/[T3oR3o] and ligand dissociation constant KRo consists of a positive cooperative change from high to low symmetry that results in "half-site reactivity". The second transition, T3oR3o right harpoon over left harpoon R3R3', with allosteric constant LoB = [T3oR3o]/[R3R3'] and ligand dissociation constant KR is a change from low to high symmetry, which is also a positive cooperative process. Treatment of the two transitions as concerted and interconnected processes allows derivation of an equation for the fraction of R-state. Using this equation, the effects of changes in the four physical parameters, LoA, LoB, KR, and KRo, on the ligand binding properties of the insulin hexamer are quantitatively described.

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

“…These observations suggest that some level of "preorganization" arises by generation of asymmetry upon binding the first ligand. [93][94][95][96] A corollary of this model is that ligand-ligand cooperativity plays an important role in catalysis. Inextricably coupled to the 222 symmetry is the use of a "one site fits both" approach as well as the inability of the enzyme to accumulate mutations without strongly affecting either protein stability, oligomerization state or binding and catalysis.…”

Insulin and its receptor: structure, function and evolution

“…(17) It plays an important role in the pharmaceutical formulations of insulin where phenol is used as an antimicrobial agent and chloride as an isotonic agent. The basic insulin fold described above (three helices, three conserved disulfide bridges) is present in all members of the insulin peptide family (Fig.…”

Analysis of Insulin Allostery in Solution and Solid State With FTIR

“…The binding of phenol to the insulin hexamer in this study has been described by a hyperbolic one-site binding model which gives the best fit to the data, and not by a sigmoidal binding curve corresponding to a cooperative binding of phenol. [5][6][7][8][9][10] The lack of cooperative binding can be explained by the presence of 50 mM NaCl, which acts as a positive heterotropic ligand to the hydrophobic binding site. Thus, the presence of chloride ions decreases the cooperativity of the system and increases the affinity between insulin and phenol.…”

“…[2][3][4] The hexameric structure acts as an allosteric unit and is modulated by homotropic and heterotropic interactions. [5][6][7][8][9][10] Three global conformations of the insulin hexamer have been identified by X-ray crystallography 2,[11][12][13][14][15] and in solution [16][17][18] and are designated T 6 , R 3 T 3 and R 6 . The structural transition from T to R form involves rearrangement of the first eight amino acid residues in the B-chain, from an extended conformation in the T state to a-helix in the R state.…”