The Structure of Insulin
The insulin molecule has served as a model for multitudes of studies on the fundamental structure and properties of proteins. It was the first protein to have its amino acid sequence sequenced, in 1955 by Fred Sanger (Sanger 1988), earning him a Nobel prize in 1958. It was also the first peptide hormone, circulating in minute amounts, to be measured by radioimmunoassay (Berson and Yallow 1961), earning Yalow a Novel Prize in 1977. The pathway behind the biosynthesis of insulin in pancreatic beta cells, specifically as a proinsulin precursor, was determined by Don Steiner in 1967 (Steiner and James 1992). The three-dimensional structure of insulin was ultimately solved by Dorothy Crowfoot Hodgkin and colleagues in 1969, using X-ray crystallographic methods (Adams et al. 1969). It was also the first protein to be synthesized in microorganisms by recombinant DNA technology in the late 1970s. This supported the design of insulin analogues in order to optimize the molecule's pharmacodynamic profile for therapeutic purposes. As a result, recombinant insulin has replaced purified insulin for therapeutic purposes. Here, we briefly discuss the structural characteristics and structure-function relationships of insulin.
The structure of insulin
Panel 1. Basic structural parameters of mature human insulin . The A chain of human insulin is shown in orange and the B chain is shown in blue ...
Circulating, and biologically active, insulin is monomeric. It is composed of two polypeptide chains: chain A has 21 amino acids and chain B has 30 amino acids (in humans). Two disulfide bridges (residues A7 to B7, and A20 to B19) covalently tether the chains, and chain A contains an internal disulfide bridge (residues A6 to A11). Notably, the positions of these three disulfide bonds are invariant in mammalian forms of insulin. The amino acid sequence of both polypeptide chains and disulfide bridge positions are shown in panel 1. At micromolar concentrations, insulin dimerizes, and in the presence of zinc, it further associates into hexamers.
The A chain has an amino-terminal helix (A1-A8) linked to an antiparallel carboxy-terminal helix (A12-A20). The B chain has a central helix (B8-B19), flanked by extended amino- and carboxy-terminal strands. This arrangement is called the "T" conformation. In the 2-Zn structure, solved by Hodgkins and colleagues (Adams et al. 1969), all six monomers are in the T conformation (T6). An alternate "R" conformation exists where the B chain helix extends from the N-terminus (B1-B19, versus B8-B19). In the 4-Zn hexamer, three of the monomers are in the T form and three are in the R form (R3T3) , as a result of a high chloride concentration (Bentley et al. 1976). An R6 form exists in phenol-containing crystals (Derewenda et al. 1989) and in solution (Chang et al. 1997). An allosteric equilibrium controls the T-R transition (Bloom et al. 1997), which plays an important role in the formulation of therapeutic insulin where chloride is used as an isotonic agent and phenol is used as an antimicrobial agent.
Panel 2. Quaternary arrangement of insulin. Residue numbers are denoted for the helical regions of chains A (orange) and B (blue)...
The hormone has a compact three-dimensional structure, consisting of three helices and three conserved disulfide bridges (Panel 2). This basic fold is present in all members of the insulin peptide family, despite divergent sequences. A cluster of hydrophobic residues that form the core of the small protein contributes to protein stability. This is further enhanced by constraint of the polypeptide backbone by the disulfide bridges. Surrounding its core, the monomer has two extensive nonpolar surfaces. The first is flat and mostly aromatic, and is buried upon dimer formation contributing to an antiparallel beta sheet structure. The other surface is more extensive and is buried upon hexamer formation. Interestingly, insulin uses the same surfaces for binding to its cognate receptor that it does for self-assembly.