Molecular Mechanism of Dimerization of Bowman-Birk Inhibitors

Abstract

Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, is a crop native to Southeast Asia and tropical Africa. The seeds contain multiple forms of Bowman-Birk type inhibitors. The major inhibitor HGI-III, from the native seed with 76 amino acid residues exists as a dimer. The amino acid sequence of three isoforms of Bowman-Birk inhibitor from germinated horsegram, designated as HGGI-I, HGGI-II, and HGGI-III, have been obtained by sequential Edman analyses of the pyridylethylated inhibitors and peptides derived therefrom by enzymatic and chemical cleavage. The HGGIs are monomers, comprising of 66, 65, and 60 amino acid residues, respectively. HGGI-III from the germinated seed differs from the native seed inhibitor in the physiological deletion of a dodecapeptide at the amino terminus and a tetrapeptide, -SHDD, at the carboxyl terminus. The study of the state of association of HGI-III, by size-exclusion chromatography and SDS-PAGE in the presence of 1 mM ZnCl2, has revealed the role of charged interactions in the monomer <--> dimer equilibria. Chemical modification studies of Lys and Arg have confirmed the role of charge interactions in the above equilibria. These results support the premise that a unique interaction, which stabilizes the dimer, is the cause of self-association in the inhibitors. This interaction in HGI-III involves the epsilon-amino group of the Lys24 (P1 residue) at the first reactive site of one monomer and the carboxyl of an Asp86 at the carboxyl terminus of the second monomer. Identification of the role of these individual amino acids in the structure and stability of the dimer was accomplished by chemical modifications, multiple sequence alignment of legume Bowman-Birk inhibitors, and homology modeling. The state of association may also influence the physiological and functional role of these inhibitors.