Insulin hexamers, structures

The crystal structure of this protein has been shown to depend on the salt solution from which it is crystallized.45 When crystallized from (NH4)2S04, the insulin hexamer is held together in part by two Zn2+ ions. These can be visualized as being at either end of a cylinder, and each Zn2 has as a ligand one histidine imidazole nitrogen atom from one of three chains. Thus a histidine (his B.10) of each of the six chains is coordinated to Zn2+, three at each Zn2+. The Zn2+ ions occupy octahedral (trigonally distorted) sites overall. 

Solution structures of the R-6 human insulin hexamer. Biochemistry, 36, 9409-9422. 

Figure 3 The structural levels of proteins, exemplified by human insulin in the T6 form. (A) Primary structure residues 15-18 of human insulin B-chain, shown as sticks. (B) Secondary structure residues 8-20 of the B-chain form an a-helix, here depicted as a superposition of sticks, and a cartoon-representation. (C) Tertiary structure insulin A- and B-chains fold up to a monomer, which is assumed to be the active form, binding to the insulin receptor. Insulin can exist in different oligomeric forms, depending on formulation and protein concentration. (D) The Zn -stabilized hexamer form is shown. 2 Zn ions are bound per insulin hexamer (only one Zn2" "-ion is visible in this view). The hexamer is a trimer of dimmers. Figure based on pdb-file IMSO, produced in Pymol. Source Bente Vestergaard, Biostructural Research, Faculty of Pharmaceutical Sciences, University of Copenhagen.

In addition to the above-mentioned excipients and functions, preservation of formulations in multiple dose containers is also required. Therefore, antimicrobial preservatives such as phenol, methyl-, and propylparabens are added. Apart from their formulation function the excipients also interact specifically with the protein and can thereby cause alterations in its function and stability (7). For example, phenol has an influence on the stability and conformation of the insulin hexamer. The addition of phenol shifts the structure from the less stable T3 to the more stable R6 (71). 

Insulin is a peptide hormone (MW = 5734) it contains an A peptide of 21 amino acids and a B subunit of 30 amino acids, linked by one intrasubunit and two intersubunit disulfide bonds (Figure 60-1). The two chains of insulin form a highly ordered structure with a-helical regions in each of the chains. In solution, insulin can exist as a monomer, dimer, or hexamer. Two molecules ofZn are coordinated in the hexamer, and this form of insulin presumably is stored in the /3 cell granules. Insulin hexamers also comprise most of the highly concentrated preparations used for therapy. As the concentration falls to physiological levels (nM), the hormone dissociates into monomers, which likely are the biologically active form. 

Smith GD, Swenson DC, Dodson EJ, et al. Structural stability in 4-zinc human insulin hexamer. Proc Natl Acad Sci USA 1984 81 7093-7097. 

Fig. 2. Three-dimensional structural representations for zinc metall-oproteins. Comparison of the zinc ion-protein bonding interactions for zinc requiring enzymes (A—C) with the zinc-insulin hexamer (D, E). (A) Human carbonic anhydrase C, redrawn from Ref. (47) with permission. (B) Bovine carboxypeptidase Ay, redrawn from Ref. 30) with permission. (C) Bacillus thermoprotedyticus thermolysin, redrawn from Ref. 45) with permission. (D) and (E) Porcine Zn-insulin hexamer, taken from Ref. 48) with permission. The composite electron density maps in (D) and (E) show that each of the two zinc atoms present in the hexamer is within inner sphere bonding distance of three solvent molecules and three histidyl imidazolyl groups in an octahedral array about the metal ion. The position of one of the three equivalently positioned solvent molecules is indicated in (D). The electron density map in (E) shows the relative orientations of the three histidyl residues (His-BlO). (The atomic positions of one of the three equivalent histidyl groups are shown)...

X-ray, circular dichroism, and centrifuge studies have demonstrated that most mammalian and fish insulins form zinc insulin hexamers similar to those of porcine insulin (Blundell et al, 1972 Blundell and Wood, 1975), with the exception of hagfish insulin, which produces only dimers (Cutfield et al, 1974 Peterson et al, 1974), and guinea pig (Zimmerman et al, 1972), casiragua (Horuk et al, 1979), and porcupine (Horuk et al, 1980) insulins, which exist only as monomers. A complete structural analysis of hagfish insulin dimers shows that, unlike porcine insulin, the two molecules of the dimer in the crystals are exactly equivalent and resemble molecule II of the asymmetric dimer of porcine insulin. This is similar to the structure of porcine insulin in solution as indicated by circular dichroism studies (Wood et al, 1975 Strickland and Mercola, 1976). 

Hormones.— Further details of the structure of zinc insulin hexamers based on a 2.8 A resolution electron-density map (see Figures 10 and 11) and the relation of the structure to the chemistry, sequence variation, and biological role of insulin, have been discussed. ... 

Simulation of the structure of the insulin hexamer consisting of six insulin monomers bound together. The amino acid chains are depicted as ribbons, except for the six histidine residues which point toward the center of the molecule. At the very center, bonded to the histidines and locking the whole structure together, is a spherical zinc ion... 

Quaternary structure. Due to non-covalent interactions, many proteins assemble to form symmetrical complexes (oligomers). The individual components of oligomeric proteins (usually 2-12) are termed subunits or monomers. Insulin also forms quaternary structures. In the blood, it is partly present as a dimer. In addition, there are also hexamers stabilized by Zn ions (light blue) (3), which represent the form in which insulin is stored in the pancreas (see p.l60). 

Figure 7-17 The structure of insulin. (A) The amino acid sequence of the A and B chains linked by disulfide bridges. (B) Sketch showing the backbone structure of the insulin molecule as revealed by X-ray analysis. The A and B chains have been labeled. Positions and orientations of aromatic side chains are also shown. (C) View of the paired N-terminal ends of the B chains in the insulin dimer. View is approximately down the pseudo-twofold axis toward the center of the hexamer. (D) Schematic drawing showing packing of six insulin molecules in the zinc-stabilized hexamer.

Insulin is able to form at least six different crystalline modifications, either zinc-free or with two or four zinc ions per hexamer. The addition of protamine and phenol also affects the structure of the crystal (for a review see Brange, 1987). 

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