Zinc–Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer – A Review

Abstract

Zinc and calcium ions play important roles in the biosynthesis and storage of insulin. Insulin biosynthesis occurs within the beta-cells of the pancreas via preproinsulin and proinsulin precursors. In the golgi apparatus, proinsulin is sequestered within Zn(2+)- and Ca(2+)-rich storage/secretory vesicles and assembled into a Zn(2+) and Ca(2+) containing hexameric species, (Zn(2+))(2)(Ca(2+))(Proin)(6). In the vesicle, (Zn(2+))(2)(Ca(2+))(Proin)(6) is converted to the insulin hexamer, (Zn(2+))(2)(Ca(2+))(In)(6), by excision of the C-peptide through the action of proteolytic enzymes. The conversion of (Zn(2+))(2)(Ca(2+))(Proin)(6)to (Zn(2+))(2)(Ca(2+))(In)(6) significantly lowers the solubility of the hexamer, causing crystallization within the vesicle. The (Zn(2+))(2)(Ca(2+))(In)(6) hexamer is an allosteric protein that undergoes ligand-mediated interconversion among three global conformation states designated T(6), T(3)R(3) and R(6). Two classes of allosteric sites have been identified; hydrophobic pockets (3 in T(3)R(3) and 6 in R(6)) that bind phenolic ligands, and anion sites (1 in T(3)R(3) and 2 in R(6)) that bind monovalent anions. The allosteric states differ widely with respect to the physical and chemical stability of the insulin subunits. Fusion of the vesicle with the plasma membrane results in the expulsion of the insulin crystals into the intercellular fluid. Dissolution of the crystals, dissociation of the hexamers to monomer and transport of monomers to the liver and other tissues then occurs via the blood stream. Insulin action then follows binding to the insulin receptors. The role of Zn(2+) in the assembly, structure, allosteric properties, and dynamic behavior of the insulin hexamer will be discussed in relation to biological function.

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

“…The maintenance of the hexameric form there, despite the lack of the entire dimer ␤-strand interface, is somehow compatible with the association of DOI into a R 6 hexamer observed in the presence of Zn 2ϩ and cyclohexanol (29). Although the T 6 3 T 3 R 3 3 R 6 dynamic transitions are rather well described by the SMB model (51)(52)(53)(54)(55)(56), the conformational events on the monomer 3 dimer 3 hexamer pathway are much less understood. Our results presented here provide further evidence that the nature of the changes on the insulin dimer interfaces (and associated other parts of the insulin molecule) is quite asymmetrical (Fig.…”

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

“…An intriguing example is the glucose-dependent acidification of insulin secretory vesicles (37). Although the exact physiological role of this acidification is unknown, it has been suggested that it may enhance the stabilization of insulin hexamers (38). The pH dependence of Zn 2ϩ transport mediated by ZnT5, as described here, provides a plausible mechanism by which an increased H ϩ electrochemical gradient across the membrane of insulin secretory vesicles will trigger enhanced Zn 2ϩ sequestration, followed by an accelerated rate of insulin hexamer formation.…”

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

“…1E), so named by analogy to hemoglobin. Although the functional significance of the T 3 R transition is not well understood (46,57), studies of insulin analogs have suggested that such allostery exploits sites of conformational change relevant to induced fit of the hormone on receptor binding (54,58).…”

“…7). Although there were no zinc ions in the running buffer, past studies have indicated that its release from hexameric complexes is dependent on their disassembly (45,46).…”