Insulin dimer, structure

Insulin was first identified as an anti-diabetic factor in 1921, and was introduced clinically the following year. Its complete amino acid sequence was determined in 1951. Although mature insulin is a dimeric structure, it is synthesized as a single polypeptide precursor, i.e. preproinsulin. This 108 amino acid polypeptide contains a 23 amino acid signal sequence at its amino terminal end. This guides it through the endoplasmic reticulum membrane, where the signal sequence is removed by a specific peptidase. 

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.

Baker et al. (1987, 1988) described a 1.5 A resoludon structure of 2Zn-insulin. They located 282 of the estimated 285 waters per insulin dimer in the crystal. These were distributed among 349 sites 217 of occupancy 1.0 126 of occupancy 0.5 five of occupancy 0.33 and one of occupancy 0.25. There was evidence for ordered water at a distance 8 A from the protein surface. Nearly 100 waters were bonded only to other waters. The extent of order of the water, judged by B values, increased with an increased number of interactions with the protein. The waters bonded to the protein act as anchors for chains of less well-ordered waters, which are often linked by threads of density, possibly representing paths along which the less-ordered waters are found. There were alternate water positions, sometimes collected into networks of partially occupied sites. Cyclic water structures were found. The protein—water contacts showed preferred geometries. Baker et al. (1988) gave particularly elegant descriptions of the crystal water. 

Figure 2.11 In situ STM of human insulin on single-crystal Au electrode surfaces [1 70], At left is a structural representation of the expected dominating insulin dimer form (PDB 1 B9E). The A- and B-chains and the three disulfide groups in each monomer (blue, red,...

The A chains in each molecule of the 4Zn insulin dimer are quite similar to each other in conformation, although not in disposition to the B chains. In molecule I the A chain has moved away from the B chain (Fig. 3) as a result of the movement of the A7-B7 disulfide link, which causes a cleft to appear in this molecule. This variation in structure of molecule I, particularly the formation of the cleft, does not seem to impair biological activity of 4Zn insulin preparations there is good evidence that on dissociation of the 4Zn hexamers the monomeric conformations are closely similar to that of molecule II in the two hexameric forms. Indeed, it has been suggested that the flexible character of insulin could be an important factor in generating biological response. 

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). 

Histamine gives a stable complex with Co +, as it gives the same sort of complex with Cu + (see above). It is interesting to link this result with the fact that insulin dimerizes through the presence in its structure of histidine and through the participation of the zinc ion Zn +, which is divalent. 

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). 

The van der Waals model of monomeric insulin (1) once again shows the wedge-shaped tertiary structure formed by the two chains together. In the second model (3, bottom), the side chains of polar amino acids are shown in blue, while apolar residues are yellow or pink. This model emphasizes the importance of the hydrophobic effect for protein folding (see p. 74). In insulin as well, most hydrophobic side chains are located on the inside of the molecule, while the hydrophilic residues are located on the surface. Apparently in contradiction to this rule, several apolar side chains (pink) are found on the surface. However, all of these residues are involved in hydrophobic interactions that stabilize the dimeric and hexameric forms of insulin. 

Recent studies also point to the existence of a fourth receptor species. This appears to be a hybrid structure, composed of an insulin receptor a-/l dimer crosslinked to an IGF-1 receptor oc-fi dimer. Although this receptor type displays a marked reduction in its affinity for insulin. 

Insulin Lispro was the first recombinant fast-acting insulin analogue to gain marketing approval (Table 8.3). It displays an amino acid sequence identical to native human insulin, with one alteration — an inversion of the natural proline lysine sequence found at positions 28 and 29 of the insulin jS-chain. This simple alteration significantly decreased the propensity of individual insulin molecules to self-associate when stored at therapeutic dose concentrations. The dimerization constant for Insulin Lispro is 300 times lower than that exhibited by unmodified human insulin. Structurally, this appears to occur as the change in sequence disrupts the formation of inter-chain hydrophobic interactions critical to self-association. 

Structure-Activity Correlations. This detailed knowledge of the three-dimensional structure of insulin led to the recognition that its biological activity resides in an area of the molecule rather than in specific amino acid residues, just as dimerization and further association of the molecule also depend on an intact spatial structure. The foregoing concept is corroborated by structural modifications of the hormone. The last three amino acids of the B chain can be removed without a loss of activity, but cleavage of the C-terminal of the A chain (Asn ) results in a total loss of activity. Amino acids can be replaced inside the chains only if such substitution does not change the overall geometry of the molecule. The structure-activity relationships of insulin derivatives are inconsistent and not always comparable. 

FIGURE 12-7 Activation of the insulin-receptor Tyr kinase by autophosphorylation. (a) In the inactive form of the Tyr kinase domain (PDB ID 11RK), the activation loop (blue) sits in the active site, and none of the critical Tyr residues (black and red ball-and-stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between Tyr1162 and Asp"32, (b) When insulin binds to the a chains of insulin receptors, the Tyr kinase of each /3 subunit of the dimer phosphorylates three Tyr residues (Tyr"58, Tyr"62, and... 

The insulin receptor is the prototype for a number of receptor enzymes with a similar structure and receptor Tyr kinase activity. The receptors for epidermal growth factor and platelet-derived growth factor, for example, have structural and sequence similarities to the insulin receptor, and both have a protein Tyr kinase activity that phosphorylates IRS-1. Many of these receptors dimerize after binding ligand the insulin receptor is already a dimer before insulin binds. The binding of adaptor proteins such as Grb2 to (P) Tyr residues is a common mechanism for promoting protein-protein interactions, a subject to which we return in Section 12.5. 

Another problem with small models is that molecules from the solution (e.g. water) may come in and stabilise tetragonal structures and higher coordination numbers [224]. It is illustrative that very few inorganic con5)lexes reproduce the properties of the blue copper proteins [66,67], whereas typical blue-copper sites have been constructed in several proteins and peptides by metal substitution, e.g. insulin, alcohol dehydrogenase, and superoxide dismutase [66]. This shows that the problem is more related to protection from water and dimer formation than to strain. 

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