Structure of the bifunctional inhibitor of trypsin and alpha-amylase from ragi seeds at 2.2 angstrom resolution

The crystal structure of a bifunctional inhibitor of alpha-amylase and tryp sin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertne ri) has been determined by X-ray diffraction at 2.2 Angstrom resolution. Th e inhibitor consists of 122 amino acids, with five disulfide bridges, and b elongs to the plant alpha-amylase/trypsin inhibitor family. The crystals we re grown by the microdialysis method using ammonium sulfate as a precipitat ing agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 Angstrom resolution determined previously. It has been refi ned to an R factor of 21.9%. The structure shows an r.m.s. deviation for C- alpha atoms of 2.0 Angstrom compared with its own NMR structure, whereas th e corresponding value compared with CHFI is found to be 1.4 Angstrom. The r .m.s. difference for C-alpha atoms when compared with the same protein in t he structure of the complex with alpha-amylase is 0.7 Angstrom. The conform ations of trypsin-binding loop and the alpha-amylase-binding N-terminal reg ion were also found to be similar in the crystal structures of native RATI and its complex with alpha-amylase. These regions differed considerably in the NMR structure.