, Volume 18, Issue 4, pp 295–303 | Cite as

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

  • Michael F. DunnEmail author


Zinc and calcium ions play important roles in the biosynthesis and storage of insulin. Insulin biosynthesis occurs within the β-cells of the pancreas via preproinsulin and proinsulin precursors. In the golgi apparatus, proinsulin is sequestered within Zn2+- and Ca2+-rich storage/secretory vesicles and assembled into a Zn2+ and Ca2+ containing hexameric species, (Zn2+)2(Ca2+)(Proin)6. In the vesicle, (Zn2+)2(Ca2+)(Proin)6 is converted to the insulin hexamer, (Zn2+)2(Ca2+)(In)6, by excision of the C-peptide through the action of proteolytic enzymes. The conversion of (Zn2+)2(Ca2+)(Proin)6to (Zn2+)2(Ca2+)(In)6 significantly lowers the solubility of the hexamer, causing crystallization within the vesicle. The (Zn2+)2(Ca2+)(In)6 hexamer is an allosteric protein that undergoes ligand-mediated interconversion among three global conformation states designated T6, T3R3 and R6. Two classes of allosteric sites have been identified; hydrophobic pockets (3 in T3R3 and 6 in R6) that bind phenolic ligands, and anion sites (1 in T3R3 and 2 in R6) 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 Zn2+ in the assembly, structure, allosteric properties, and dynamic behavior of the insulin hexamer will be discussed in relation to biological function.

Key words

allosteric transitions calcium hexamer insulin zinc 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ashcroft, FM, Ashcroft, SJH 1992Insulin Molecular Biology to PathologyOxford University PressNew York592Google Scholar
  2. Baker, EN, Blundell, TL, Cutfield, JF,  et al. 1988The structure of 2Zn pig insulin crystals at 1.5 Å resolutionPhil Trans Roy Soc (London) B319369456Google Scholar
  3. Banting, FG, Best, CH 1922Pancreatic extractsJ Lab Clin Med7464472Google Scholar
  4. Bloom, CR, Choi, WE, Brzovic, PS, Ha, JJ, Huang, ST, Kaarsholm, NC, Dunn, MF 1995Ligand binding to wild-type and E-B13Q mutant insulins; A three-state allosteric model system showing half-site reactivityJ Mol Biol245324330CrossRefPubMedGoogle Scholar
  5. Bloom, CR, Heymann, R, Kaarsholm, NC, Dunn, MF 1997aBinding of 2,6- and 2,7-dihydroxynaphthalene to wild-type and E-B13Q insulins: Dynamic, equilibrium and molecular modeling investigationsBiochemistry361274612758CrossRefGoogle Scholar
  6. Bloom, CR, Kaarsholm, NC, Ha, JJ, Dunn, MF 1997bHalf-site reactivity, negative cooperativity, and positive cooperativity: Quantitative considerations of a plausible modelBiochemistry361275912765CrossRefGoogle Scholar
  7. Bloom, CR, Wu, N, Dunn, A, Kaarsholm, NC, Dunn, MF 1998Comparison of the allosteric properties of the Co(II) and Zn(II) substituted insulin hexamersBiochemistry371093710944CrossRefPubMedGoogle Scholar
  8. Blundell, T, Dodson, G, Hodgkin, D, Mercola, D 1972Insulin: The structure in the crystal and its reflection in chemistry and biologyAdv Protein Chem26279402Google Scholar
  9. Brader, ML, Dunn, MF 1991Insulin hexamers: New conformations and applicationsTrends Biochem Sci16341345CrossRefPubMedGoogle Scholar
  10. Brader, ML, Kaarsholm, NC, Harnung, SE, Dunn, MF 1997Ligand perturbation on a pseudotetrahedral Co(II)(His)3L site. A magnetic circular dichroism study of the Co(II)-substituted insulin hexamerJ Biol Chem27210881094CrossRefPubMedGoogle Scholar
  11. Brader, ML, Kaarsholm, NC, Lee, RW-K, Dunn, MF 1991Characterization of the R-state insulin hexamer and its derivatives. The hexamer is stabilized by heterotropic ligand binding interactionsBiochemistry3066366645CrossRefPubMedGoogle Scholar
  12. Brzovic, PS, Choi, WE, Borchardt, D, Kaarsholm, NC, Dunn, MF 1994Structural asymmetry and half-site reactivity in the T to R allosteric transition of the insulin hexamerBiochemistry331305713069CrossRefPubMedGoogle Scholar
  13. Choi, WE, Brader, ML, Aguilar, V, Kaarsholm, NC, Dunn, MF 1993The allosteric transition of the insulin hexamer is modulated by homotropic and heterotropic interactionsBiochemistry321163811645CrossRefPubMedGoogle Scholar
  14. Ciszak, E, Smith, GD 1994Crystallographic evidence for dual coordination around zinc in the T3R3 human insulin hexamerBiochemistry3315121517CrossRefPubMedGoogle Scholar
  15. Coffman, FD, Dunn, MF 1988Insulin–metal ion interactions. The binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamersBiochemistry2761796187CrossRefPubMedGoogle Scholar
  16. Derewenda, U, Derewenda, Z, Dodson, EJ,  et al. 1989Phenol stabilizes more helix in a new symmetrical zinc insulin hexamerNature338594596CrossRefPubMedGoogle Scholar
  17. Hill, CP, Dauter, Z, Dodson, EJ, Dodson, GG, Dunn, MF 1991X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamerBiochemistry30919924Google Scholar
  18. Howell, SL, Montague, W, Tyhurst, M 1975Ca distribution in islets of LangerhansJ Cell Sci19395409PubMedGoogle Scholar
  19. Huang, ST, Choi, WE, Bloom, CR, Leuenberger, M, Dunn, MF 1997Carboxylate ions are strong allosteric ligands for the HisB10 sites of the R-state insulin hexamerBiochemistry3698789888CrossRefPubMedGoogle Scholar
  20. Kaarsholm, NC, Ko, H-C, Dunn, MF 1989Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin and mini-proinsulinBiochemistry2844274435CrossRefPubMedGoogle Scholar
  21. Kadima, W, Roy, M, Lee, RW-K, Kaarsholm, NC, Dunn, MF 1992Mechanisms of stabilization of the insulin hexamer through allosteric ligand interactionsJ Biol Chem26789638970PubMedGoogle Scholar
  22. Olsen, HB, Leuenberger-Fisher, MR, Kadima, W, Borchardt, D, Kaarsholm, NC, Dunn, MF 2003Structural signatures of the complex formed between 3-nitro-4-hydroxybenzoate and the Zn(II)-substituted R6 insulin hexamerProtein Sci1219021913CrossRefPubMedGoogle Scholar
  23. Rahuel-Clermont, S, French, CA, Chou, CI, Kaarsholm, NC, Dunn, MF 1997Mechanisms of stabilization of the insulin hexamer through allosteric ligand interactionsBiochemistry3658375845CrossRefPubMedGoogle Scholar
  24. Roy, M, Brader, ML, Lee, RW-K, Kaarsholm, NC, Hansen, J, Dunn, MF 1989Spectroscopic signatures of the T to R conformational transition in the insulin hexamerJ Biol Chem2641908119085PubMedGoogle Scholar
  25. Seydoux, F, Malhotra, OP, Bernhard, SA 1974Half-site reactivityCRC Crit Rev Biochem2227257PubMedGoogle Scholar
  26. Smith, GD, Dodson, GG 1992aThe structure of a rhombohedral R6 insulin hexamer that binds phenolBiopolymers3217491756Google Scholar
  27. Smith, GD, Dodson, GG 1992bStructure of a rhombohedral R6 insulin/phenol complexProteins: Struct Funct Genet14401408CrossRefGoogle Scholar
  28. Smith, GD, Ciszak, E, Magrum, LA, Pangborn, WA, Blessing, RH 2000R6 hexameric insulin complexed with m-cresol or resorcinolActa CrystD5615411548Google Scholar
  29. Storm, MC, Dunn, MF 1985The Glu(B13) carboxylates of the insulin hexamer form a cage for Cd2+ and Ca2+ ionsBiochemistry2417491756CrossRefPubMedGoogle Scholar
  30. Sudmeier, JL, Bell, SJ, Storm, MC, Dunn, MF 1981Cadmium-113 nuclear magnetic resonance studies of bovine insulin: Two-zinc insulin hexamer specifically binds calciumScience212560562PubMedGoogle Scholar
  31. Whittingham, JL, Chaudhuri, S, Dodson, EJ, Moody, PCE, Dodson, GG 1995X-ray crystallographic studies on hexameric insulins in the presence of helix-stabilizing agents, thiocyanate, methylparaben, and phenolBiochemistry341555315563CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of CaliforniaRiversideUSA

Personalised recommendations