CRYSTALLOGRAPHIC EVIDENCE FOR DUAL COORDINATION AROUND ZINC IN THE T(3)R(3) HUMAN INSULIN HEXAMER

The T(3)R(3) human insulin hexamer (T and R referring to extended and alpha-helical conformations, respectively, of the first eight residues of the B-chain), complexed to two zinc ions, crystallizes in space group R3 with hexagonal cell constants a = 80.64 and c = 37.78 Angstrom. The structure has been refined to a residual of 0.172 using 9225 independent data to 1.6-Angstrom resolution. The asymmetric unit consists of a TR dimer, and the insulin hexamer is generated by the action of the crystallographic 3-fold axis. The conformation of one insulin trimer is nearly identical to that of the T-6 hexamer, while the other trimer approximates that of the R(6) hexamer, except for the three N-terminal B-chain residues that adopt an extended rather than an alpha-helical conformation. Each of the two zinc ions, which lie on the crystallographic 3-fold axis and exhibit two different, disordered coordination geometries, is coordinated by the imidazole groups of three symmetry-related B10 histidine residues. The coordination sphere of the zinc in the T-3 trimer is either tetrahedral, with the fourth site filled by a chloride ion, or octahedral, completed by three water molecules. The coordination of the zinc in the 12-Angstrom narrow channel in the R(3) trimer is tetrahedral, with either a second chloride ion or a water molecule completing the coordination sphere. The putative off-axial zinc binding sites that result from the T-->R transition of monomer II do not contain zinc ion, but instead are filled with clusters of ordered water molecules. The observation that the T-state trimer contains zinc in both tetrahedral and octahedral geometries has important implications for the interpretation of spectroscopic results.