Structural consequences of the B5 histidine -> tyrosine mutation in human insulin characterized by x-ray crystallography and conformational analysis

The addition of phenols to hexameric insulin solutions produces a particula rly stable hexamer, resulting from a rearrangement in which residues B1-B8 change from an extended conformation (T-state) to form an alpha-helix (R-st ate). The R-state is, in part, stabilized by nonpolar interactions between the phenolic molecule and residue B5 His at the dimer-dimer interface. The B5 His --> Tyr mutant human insulin was constructed to see if the tyrosine side chain would mimic the effect of phenol binding in the hexamer and indu ce the R-state. In partial support of this hypothesis, the molecule crystal lized as a half-helical hexamer (T3R3) in conditions that conventionally pr omote the fully nonhelical (T-6) form. As expected, in the presence of phen ol or resorcinol, the B5 Tyr hexamers adopt the fully helical (R-6) conform ation. Molecular modeling calculations were performed to investigate the co nformational preference of the T-state B5 Tyr side chain in the T3R3 form, this side chain being associated with structural perturbations of the A7-A1 0 loop in an adjacent hexamer. For an isolated dimer, several different ori entations of the side chain were found, which were close in energy and read ily interconvertible. In the crystal environment only one of these conforma tions remains low in energy; this conformation corresponds to that observed in the crystal structure. This suggests that packing constraints around re sidue B5 Tyr result in the observed structural rearrangements. Thus, rather than promoting the R-state in a manner analogous to phenol, the mutation a ppears to destabilize the T-state. These studies highlight the role of B5 H is in determining hexamer conformation and in mediating crystal packing int eractions, properties that are likely be important in vivo.