Zinc insulin hexamers

Proinsulin is proteolytically processed in the coated secretory granules, yielding mature insulin and a 34-amino acid connecting peptide (C peptide, Figure 11.1). The C peptide is further proteolytically modified by removal of a dipeptide from each of its ends. The secretory granules thus contain low levels of proinsulin, C peptide and proteases, in addition to insulin itself. The insulin is stored in the form of a characteristic zinc-insulin hexamer, consisting of six molecules of insulin stabilized by two zinc atoms. 

The concentration of insulin present in soluble insulin preparations (i.e. fast-acting insulins), is much higher (approximately 1 x KT2 3 mol I ). At this concentration, the soluble insulin exists as a mixture of monomer, dimer, tetramer and zinc-insulin hexamer. These insulin complexes have to dissociate in order to be absorbed from the injection site into the blood, which slows down the onset of hormone action. 

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. ... 

Although insulin was first crystallized in 1926, the factors promoting crystal growth were poorly understood and yielded inconsistent results. It was almost 10 years later when researchers discovered that the addition of zinc to a crude extract promoted reproducible crystallization (zinc addition yields a characteristic rhombohedral crystal, the basic crystal unit being the insulin hexamer, stabilized by the two zinc atoms). 

The same transformation has recently been examined by nmr in solution.46 In zinc sulfate solution, with two zinc atoms per hexamer, a certain nmr spectrum is observed. Addition of excess zinc and of chloride, iodide, or particularly thiocyanate but not sulfate converts this to a different spectrum. Detailed study suggests that this is of the six-zinc insulin, which is in equilibrium with the two-zinc form, but in slow exchange. 

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.

Figure 9.19 Suggested scheme of events after the s.c. administration of soluble human insulin the concentration of hexa-meric zinc-insulin, which is the predominant form of insulin in soluble insulin (40 Unit or 100 Unit - i.e. 0.6 mmol dm ), decreases as diffusion of insulin occurs and os a result the hexamer dissociates into smaller units to achieve monomeric insulin requires a 1000-fold dilution. The importance of the association state is that the larger species have more difficulty dispersing and passing through the capillary membrane.

The switch in position of amino acids in lispro does not affect the action of this synthetic insulin on cells because it is not in a critical invariant region, but it does affect the ability of insulin to bind zinc. Normally, human insulin is secreted from the pancreas as a zinc hexa-mer in which six insulin molecules are bound to the zinc atom. When zinc insulin is injected, the binding to zinc slows the absorption from the subcutaneous (under the skin) injection site. Lispro cannot bind zinc to form a hexamer, and thus it is absorbed much more quickly than other insulins. 

Only the insulin monomer is able to interaot with insulin receptors, and native insulin exists as a monomer at low, physiologioal oonoentrations (<0.1 pM). Insulin dimerizes at the higher oonoentrations (0.6 mM) found in pharmaceutioal preparations, and at neutral pH in the presence of zinc ions, hexamers form (34). These zino-associated hexamers also are the storage form of insulin in p cells. At concentrations greater than 0.2 mM, hexamers form even in the absence of zino ions. 

The importance of zinc ions for stabilizing insulin preparations has been known since the first reported crystallization of insulin in the presenoe of zino ions in 1934 (36). Suspensions of zinc insulin were used at that time. Presently, all pharmaceutical preparations are either solutions of zino insulin or suspensions of insoluble forms of zino insulin. A longer-acting and more stable form of insulin is protamine zinc insulin, which is prepared by precipitating insulin in the presence of zinc ions and protamine, a basic protein. This precipitate is known to contain two zinc ions per insulin hexamer. A somewhat shorter-acting and more useful preparation is neutral protamine Hagedorn (NPH) insulin, which includes m-cresol... 

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. 

Figure 1. Insulin monomers (a) associated through hydrophobic interactions and as antiparallel i -pleated sheet to dimers (b) and, in the presence of zinc, to 2Zn insulin hexamers (c). View along the crystallographic threefold axis. From Blundell et al. (1972).

Figure 4. (a) A comparison of the 2Zn insulin and 4Zn insulin hexamers viewed down the threefold axis. Only a-carbon atoms, histidine side chains, and zinc ions are shown. The local twofold axis of the dimers is drawn in bold lines, (b) View identical to that of (a) comparing the molecule I B chains of 2Zn insulin and 4Zn insulin note the changed conformation and zinc coordination, (c) The B chains of molecules I and II viewed from an equivalent direction. From Bentley et al. (1976). 

M.A. (2010) Supramolecular protein engineering design of zinc-stapled insulin hexamers as a long acting depot. Journal of Biological Chemistry, 285, 11755-11759. 

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... 

FIGURE 3.8. Insulin hexamer connected by zinc ions (I. Yonemoto). 

Generally, insulin forms a hexamer with zinc. The insulin association resulted from the zinc content in the insulin [22]. However, without zinc, insulin formed various association states such as monomer, dimer, hexamer, and aggregate. It is thought that insulin without zinc formed the aggregation state inside the gel. The aggregated insulin may not diffuse fast from the ReGel formulation, which presented a slower release (60% after 15 days). Insulin with 0.2 wt% zinc formed the hexameric state. The release profile of the insulin with... 

The insulin monomer is of 6000 molecular weight, and either the monomer or the dimer is the active hormone. Thus in fact the zinc that binds the molecule into hexamers is not of physiological interest. What is... 

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