A NOVEL COMPLEX OF A PHENOLIC DERIVATIVE WITH INSULIN - STRUCTURAL FEATURES RELATED TO THE T-]R TRANSITION

The structure of a symmetric T(3)R(3)(f) insulin hexamer, complexed wi th 4-hydroxybenzamide, has been determined using X-ray crystallographi c techniques. Data were measured from six crystals grown in microgravi ty to a resolution of 1.4 Angstrom and the structure has been refined including the contributions from hydrogen atoms. The crystals are isom orphous with T(3)R(3)(f) complexes of phenolic derivatives as well as with uncomplexed forms. Unlike the structures of complexes with phenol , m-cresol, resorcinol, 4'-hydroxyacetanilide, and methylparaben, whic h bind one phenolic derivative molecule per R- or R(f)-state monomer, two molecules of 4-hydroxybenzamide are bound by each R(f)-state monom er. The presence of the second guest molecule results in an extensive hydrogen bonding network, mediated by water molecules, between the T- and R(f)-state trimers and adds stability to the formation of the hexa mer. The only access to these second sites is through three symmetry-r elated, narrow channels that originate on the surface of the T-state t rimer. Although the conformation of the backbone atoms of the monomers is nearly identical to that of other T(3)R(3)(f) hexamers, significan t changes are observed in the conformations of side chains in the vici nity of the second binding site. The side chain of the T-state A11 Cys residue, which forms a disulfide bond to A6 Cys in the same monomer, is observed in two discrete conformations; two discrete conformations are also present for the entire A8 Thr residue in the R(f)-state monom er. A procedure is also described for an alternate method of interfram e scaling and merging intensity data from an image plate detector.