1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site and the Zn2+-Ca2+ derivative (In)6√ (Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2ZN-insulin hexamer, (In)6(Zn2+)2. The authors postulate that the additional thermodynamic drive provided by Ca2+ and Cd2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(n2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings