THE RELATIONSHIP BETWEEN INSULIN BIOACTIVITY AND STRUCTURE IN THE NH2-TERMINAL A-CHAIN HELIX

Studies of naturally occuring and chemically modified insulins have es tablished that the NH2-terminal helix of the A-chain is important in c onferring affinity in insulin-receptor interactions. Nevertheless, the three-dimensional structural basis for these observations has not pre viously been studied in detail. To correlate structure and function in this region of the molecule, we have used the solution structure of a n engineered monomer (GLuB1, GluB10, GluB16, GIuB27, desB30)-insulin ( 4E insulin) as a template for design of A-chain mutants associated wit h enhanced or greatly diminished affinity for the insulin receptor. In the context of 4E insulin, the employed mutants, i.e. ThrA8 --> His a nd ValA3 --> Gly, result in species with 143% and 0.1% biological acti vity, respectively, relative to human insulin. The high-resolution NMR studies reveal two well-defined structures each resembling the templa te. However, significant structural differences are evident notably in residues A2-A8 and their immediate environment. In comparison with th e template structure, the A8His mutation enhances the helical characte r of residues A2-A8. This structural change leads to additional exposu re of a hydrophobic patch mainly consisting of species invariant resid ues. In contrast, the A3Gly mutation leads to stretching and disruptio n of the A2-A8 helix and changes both the dimensions and the access to the hydrophobic patch exposed in the more active insulins. We conclud e that the mutations induce small, yet decisive structural changes tha t either mediate or inhibit the subtle conformational adjustments invo lved in the presentation of this part of the insulin pharmacophore to the receptor. (C) 1998 Academic Press.