STRUCTURAL DETERMINANTS OF THE BIFUNCTIONAL CORN HAGEMAN-FACTOR INHIBITOR - X-RAY CRYSTAL-STRUCTURE AT 1.95 ANGSTROM RESOLUTION

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13 .6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulatio n as well as alpha-amylases from several insect species. Among the pla sma proteinases, CHFI specifically inhibits Factor XIIa without affect ing the activity of other coagulation proteinases. We have isolated CH FI from corn and determined the crystallographic structure at 1.95 Ang strom resolution. Additionally, we have solved the structure of the re combinant protein produced in Escherichia coli at 2.2 Angstrom resolut ion. The two proteins are essentially identical. The proteinase bindin g loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the fam ily of 25 cereal trypsin/alpha-amylase inhibitors, we have made three- dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase in hibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a test able hypothesis for the regions of these proteins which bind alpha-amy lase. In the course of the investigations, we have found that the cere al trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-am ylase inhibitors, 2S seed storage albumins, and the lipid-transfer fam ily. Apparently, the four-helix conformation has been a successful veh icle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.