MUTATIONS AT THE DIMER, HEXAMER, AND RECEPTOR-BINDING SURFACES OF INSULIN INDEPENDENTLY AFFECT INSULIN INSULIN AND INSULIN-RECEPTOR INTERACTIONS

Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10 --> Asp, ProB28 --> Lys, LysB29 --> Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by H-1-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24 --> Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 > unsubstituted > GlyB24 > SerB24 > AlaB25 > LeuB25 > SerB25, whether the substitution was in a native human or DKP-insulin background. Such 'template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template.