Xray crystallography

When we started X-ray crystallography of soybean proteins, the crystal structures of the 11S globulin had not been reported; only those of French bean phaseolin and jack bean canavalin, 7S globulins, had been determined (24-26).

We tried to determine the crystal structures of the native and recombinant P3 by the molecular replacement method using the protein structure models of phaseolin and canavalin as a search model. As a result, we determined the three dimensional structure of P3 by molecular replacement using canavalin as a search model (27) [Figure 2.2]. The P monomers consist of aminoterminal and carboxyterminal modules which are very similar to each other and are related by a pseudodiad axis. Each module of the P monomer is subdivided into a P-barrel and an a-helix domain. The superposition of the models of the native and recombinant P monomers shows a root mean square deviation (RMSD) of 0.43-0.51 A for 343 common Ca atoms within 2.0 A. This result indicates that the N-linked glycan does not influence the final structure of P3. Four regions including N- and C-termini were not included in the models of all the recombinant and native P monomers, because electron density maps in these regions were too thin to trace the correct sequence. These are likely to be disordered on the molecular surface [Figure 2.3(a)]. Comparison of sequences between 7S globulins by Wright (28) showed conserved regions (amino acid identity is high) and variable regions (amino acid identity is low), and it was noted that five variable

Figure 2.2 Ribbon diagrams of the three dimensional structures of (a) P-conglycinin and (b) P-homotrimer. Numbers indicate dimensions. (From: Maruyama, N., M. Adachi, K. Takahashi, K. Yagasaki, M. Kohno, Y. Takenaka, E. Okuda, S. Nakagawa, B. Mikami, S. Utsumi, Eur. J. Biochem. 268:3595-3604, 2001.)
Figure 2.3 Disordered regions of soybean proteins. Disordered regions of P-conglycinin P (a) and of glycinin A1aB1b (b). The termini of the disordered regions are indicated by balls.

regions exist. In fact, there are only four variable regions, because one of them was identified by using an incorrect sequence of the a' subunit (15). Therefore, the disordered regions in the P subunit are consistent with the four variable regions. An amino acid replacement is liable to occur in variable regions, because they are disordered regions.

a3 and a'3 among the three homotrimers of P-conglycinin formed only small crystals, but acore3 and a'core3 with no extension region formed crystals suitable for X-ray crystallography. Therefore, the presence of the extension region probably perturbs the formation of crystals of good quality. We determined the three dimensional structure of a'core3 by the molecular replacement using the model of a3 (unpublished data). The scaffold of a'core3 was identical to that of P3. All a atoms of the a'core monomer and homotrimer could be superimposed on corresponding atoms of the P monomer and homotrimer with a small RMSD of 0.6 and 0.7 A, respectively. These values indicate that scaffolds of these two proteins are very similar to each other.

2.3.2.2 Glycinin

The amino acid identity between 7S and 11S globulins is only 15%, but they do exhibit partially significant identity. Therefore, it is considered that these two globulins are evolu-tionarily related. The 11S globulin has a hexameric structure, but proglycinin has a trimeric structure. In other words, there is a possibility that pro forms of 11S globulins have a structure analogous to the 7S globulin. Thus, we tried to determine the structure of proglycinin A1aB1b by molecular replacement using the model of P-conglycinin P3, canavalin, and phaseolin but failed. Finally, we successfully determined the structure of proglycinin A1aB1b by the multiple isomorphous replacement method using heavy atom derivatives (29). Our results showed that the structure of proglycinin A1aB1b is similar to that of P-conglycinin P (Figure 2.4). Superposition of the structure of proglycinin A1aB1b on that of P-conglycinin P exposes the high similarity between the proteins (Figure 2.5). A least squares fit of a pro-tomer between them produced an RMSD of 1.35 A, and that of P-barrel in a protomer between them was 1.2 A. Further, RMSDs of the comparable Ca atoms of P-barrel domains in both N- and C-terminal modules between proglycinin A1aB1b and P-conglycinin P were approximately 0.9 A, whereas those of a-helix domains in N- and C-terminal modules were 1.35 and 1.39 A, respectively. This indicates that the scaffold of proglycinin A1aB1b is very similar to that of P-conglycinin P, but that the configurations between a P-barrel and an a-helix domain in protomers are slightly different.

All six disordered regions existing in proglycinin A1aB 1b protomer are located on the molecular surface similarly to P-conglycinin [Figure 2.3(b)]. Amino acid sequences have been previously aligned among various 11S globulins from legumes and nonlegumes, and five variable regions have been proposed (14). Five of the six disordered regions correspond to the five variable regions. The only disordered region that does not correspond to the variable region is the shortest one. The second disordered region from the C-terminus is the

Figure 2.4 Ribbon diagram of the three dimensional structure of A1aB1b homotrimer. The view in the left diagram is depicted with the threefold symmetry axis running perpendicular to the paper, whereas the depiction on the right is related to the view on the left by a rotation of 90°. (From: Adachi, M., Y. Takenaka, A.B. Gidamis, B. Mikami, S. Utsumi., J. Mol. Biol. 305:291-305, 2001.)

Figure 2.4 Ribbon diagram of the three dimensional structure of A1aB1b homotrimer. The view in the left diagram is depicted with the threefold symmetry axis running perpendicular to the paper, whereas the depiction on the right is related to the view on the left by a rotation of 90°. (From: Adachi, M., Y. Takenaka, A.B. Gidamis, B. Mikami, S. Utsumi., J. Mol. Biol. 305:291-305, 2001.)

longest, and the region of A1aB1b is composed of 48 residues. This region is called the hypervariable region, because both an amino acid sequence and a number of an amino acid in this region are the most variable among the five.

The three dimensional structure of the native glycinin A3B4 homohexamer is determined by the molecular replacement using the model of proglycinin A1aB1b (Figure 2.6) (30). As a result, a trimer of a proglycinin assembles into a hexamer by stacking of faces in which the processing site for a mature form exists.

Figure 2.5 Superposition of the Ca trace of the P-conglycinin P and proglycinin A1aB1b. The Ca traces of P-conglycinin P and proglycinin A1aB1b are shown in gray and black lines, respectively.

Figure 2.6 Ribbon diagram of the three dimensional structure of mature A3B4 homohexamer. The view in the left diagram is depicted with the threefold symmetry axis running perpendicular to the paper. The depiction on the right is related to the view on the left by a rotation of 90°. Numbers indicate dimensions. (From: Adachi, M., J. Kanamori, T. Masuda, K. Yagasaki, K. Kitamura, M. Bunzo, S. Utsumi. Proc. Natl. Acad. Sci. USA, 100:7395-7400, 2003.)

Figure 2.6 Ribbon diagram of the three dimensional structure of mature A3B4 homohexamer. The view in the left diagram is depicted with the threefold symmetry axis running perpendicular to the paper. The depiction on the right is related to the view on the left by a rotation of 90°. Numbers indicate dimensions. (From: Adachi, M., J. Kanamori, T. Masuda, K. Yagasaki, K. Kitamura, M. Bunzo, S. Utsumi. Proc. Natl. Acad. Sci. USA, 100:7395-7400, 2003.)

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