Structural Determinants of the Bifunctional Corn Hageman Factor Inhibitor:  X-ray Crystal Structure at 1.95 Å Resolution^,

Abstract

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 coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family 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 inhibitor 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 testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal 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-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

“…Structural Modeling-The sequence alignment of ␣TTP, SPF, and Sec14 presented by Hendrickson and co-workers (15) was used as a guide for locally written programs to superimpose the ligand binding domains of these structures (28). These programs select residues for equivalence if the distance is within a cutoff threshold.…”

“…In MOE (33) the model was flooded with water molecules, energy minimized and dynamics calculated for up to 1 ns. Following the dynamics (CHARMm22, 300 K, at constant temperature and volume) the energy was minimized to finish the process (28). Model figures were drawn with Molscript (34) and Raster3d (35).…”

Antifungal Proteins

“…We have recently confirmed that at high trypsin/zeamatin and ␣-amylase/zeamatin molar ratios, zeamatin inhibits trypsin and insect ␣-amylase activities in vitro (152a). Other bifunctional proteins from ragi (Eleusine coracana), wheat, and barley have been isolated and characterized (9,46,133,152,160). Only a few of these proteins have been tested for in vitro antifungal activity, with zeamatin being VOL.…”

Proteinase inhibitors and their function in plants: A review

“…The main elements of RBI structure include four α-helical segments and a short antiparallel β-sheet segment [31,191]. In spite of differences in the overall structure of the molecules, RBI and corn trypsin inhibitor share the same conformation of the trypsinbinding site as other low-molecular-weight serine proteinase inhibitors [31,192].…”