Structure of the bifunctional inhibitor of trypsin and α-amylase from ragi seeds at 2.2 Å resolution

Abstract

The crystal structure of a bifunctional inhibitor of α-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2Å resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant α-amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating 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 Å resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C atoms of 2.0 Åcompared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 Å. The r.m.s. difference for C atoms when compared with the same protein in the structure of the complex with α-amylase is 0.7 Å. The conformations of trypsin-binding loop and the -amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with α-amylase. These regions differed considerably in the NMR structure.