Crystal structure of soybean 11S globulin: Glycinin A3B4 homohexamer

Abstract

Most plant seeds contain 11S globulins as major storage proteins for their nutrition. Soybean glycinin belongs to the 11S globulin family and consists of five kinds of subunits. We determined the crystal structure of a homohexamer of the glycinin A3B4 subunit at 2.1-Å resolution. The crystal structure shows that the hexamer has 32-point group symmetry formed by face-to-face stacking of two trimers. The interface buries the highly conserved interchain disulfide. Based on the structure, we propose that an ingenious face-to-face mechanism controls the hexamer formation of the 11S globulin by movement of a mobile disordered region to the side of the trimer after posttranslational processing. Electrostatic analysis of the faces suggests that the interchain disulfide-containing face has high positive potential at acidic pH, which induces dissociation of the hexamer into trimers that may be susceptible to proteinases after seed imbibition. This dissociation might result in the degradation and mobilization of 11S globulins as storage proteins in embryos during germination and seedling growth. P lants accumulate protein reserves in developing seeds to act as a sink of nitrogen, sulfur, and carbon. Most dicotyledonous plant seeds contain 7S and͞or 11S globulins and albumins as the major storage proteins in the embryo or cotyledons, whereas some cereals such as wheat, barley, and corn contain prolamins in the endosperm (1, 2). The 11S globulins are distributed more widely than the 7S globulins among plant seeds. Although the quaternary structures of the 7S and 11S globulins are different from each other (the former being a trimeric protein and the latter a hexameric protein), they are believed to be derived from a common ancestor because of the partial homologies in their amino acid sequences and limited proteolysis patterns (3). This assumption has been confirmed by x-ray crystallography of 7S globulins from kidney bean (4), jack bean (5), and soybean (6) and of the trimeric soybean 11S globulin precursor (7).In general, 11S globulins are composed of several kinds of subunits. For example, five major subunits have been identified from soybean: A1aB1b, A2B1a, A1bB2, A3B4, and A5A4B3. In developing seeds, the constituent subunits of 11S globulin are synthesized as a single polypeptide precursor, preproprotein, the signal sequence of which is removed cotranslationally. The resultant proproteins assemble into trimers of Ϸ8S in the endoplasmic reticulum. The proprotein trimers are transported from the endoplasmic reticulum to protein storage vacuoles (PSVs), where they then are cleaved to form acidic and basic polypeptides that are linked by a disulfide bond (8). Finally, the mature proteins assemble into hexamers. The protein reserves are stored in the dormant seed until its germination.Recently we determined the crystal structure of the soybean proglycinin A1aB1b homotrimer at 2.8-Å resolution (7). The protomer consists of N-and C-terminal modules that each include a jelly-roll ␤-barrel and a helix domain. However, the packa...

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

“…Unique tryptic peptides identified by MS in AT1G03880.1, AT1G03890.1 and AT5G44120.3, as well as the tryptic peptides in AT4G28520.1 in the divergent region of this locus are underlined. The binding regions involved in trimer interaction are double underlined and numbered according to those in glycinin A3B4 [7]. Bottom: alternative splicing diagram.…”

“…The At4g28520.2 and At4g28520.3 transcripts encode proteins with internal deletions; however, tryptic peptides capable of distinguishing these variants from At4g28520.1 were not detected. The protein encoded by At4g28520.3 would also lack the internal Asn↓Gly β-cleavage site, and processing at this location is thought to be required for hexamer assembly [7]. The proteins encoded by At5g44120.1 and At5g44120.2 would be truncated, with deletions of 187 and 104 amino acid residues near the N-terminus of each protein respectively.…”

“…Furthermore, hydroxyamino acids were found at the same, or immediately adjacent, locations in eight of the 14 corresponding sites in the soybean A3B4 glycinin. Consequently we were able to superimpose the locations of the phosphorylated residues on to the known three-dimensional structure of the soybean glycinin [7]. Interestingly, seven of the 14 sites mapped to regions on the interchain disulfide-containing (IE) face involved in the face-to-face interaction between trimers required for hexamer assembly ( Figure 5).…”

“…The processing appears to direct the relocalization of an acidic mobile disordered region (residues 252-325 in glycinin A3B4) from the surface of the IE face to the side of the protein. The mobile disordered region is proposed to prevent nonspecific interaction between the positively charged IE face and negatively charged IA (intrachain disulfide-containing) face of the trimers prior to hexamer formation [7]. Although a long acidic Each glycinin and cruciferin molecule consists of an α-and β-peptide linked together by intrachain (IA) and interchain (IE) disulfide bonds (orange) that denote the two faces.…”

“…Salt-soluble globulins are widely distributed in dicotyledons, monocotyledons and gymnosperms, and comprise the trimeric and predominantly glycosylated 7 S vicilins and the hexameric and generally non-glycosylated 11-12 S legumins [1]. The three-dimensional structures of several vicilin-type proteins, namely phaseolin [2], canavalin [3], β-conglycinin [4] and germin [5], and the legumin-type proglycinin A1aB1b homotrimer [6] and glycinin A3B4 homohexamer [7], have revealed that seed storage globulins share striking similarities in secondary and tertiary structure with the cupin superfamily of prokaryotic and eukaryotic proteins [8,9]. Proteins in this family have been ascribed a wide variety of functions [8,9].…”