Carbohydrate Binding, Quaternary Structure and a Novel Hydrophobic Binding Site in Two Legume Lectin Oligomers from Dolichos biflorus Thomas W. Hamelryck 1 *, Remy Loris 1 , Julie Bouckaert 1 Minh-Hoa Dao-Thi 1 , Gerard Strecker 2 , Anne Imberty 3 , Elias Fernandez 4 Lode Wyns 1 and Marilynn E. Etzler 4 1 Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituur voor Biotechnologie, Vrije Universiteit Brussel Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium 2 Universite  des Sciences et Technologies de Lille, Ba Ãtiment C9, 59655, Villeneuve D'Ascq CEDEX, France 3 CERMAV-CNRS (af®liated to the University Joseph Fourier) BP 53, F-38041 Grenoble Cedex 9, France 4 Section of Molecular and Cellular Biology, University of California, Davis CA 95616, USA The seed lectin (DBL) from the leguminous plant Dolichos bi¯orus has a unique speci®city among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipi- tation of blood group A  H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(a1-3)[Fuc(a1-2)]Gal) contain- ing pentasaccharide, and about 40 times better by the Forssman disac- charide (GalNAc(a1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL- Forssman disaccharide complex. A comparison with the binding sites of Gal-binding legume lectins indicates that the low af®nity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher af®nity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the ®rst structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an a-helix sandwiched between two monomers. In both oligomers, this a-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel qua- ternary structure, related to the quaternary structure of the DBL hetero- tetramer, and brings the number of know legume lectin dimer types to four. # 1999 Academic Press Keywords: protein-carbohydrate interactions; quaternary structure; legume lectins; cytokinins; X-ray crystallography *Corresponding author E-mail address of the corresponding author: thamelry@vub.ac.be Abbreviations used: DB58, horse gram (Dolichos bi¯orus) stem and leaf lectin; DBL, horse gram (Dolichos bi¯orus) seed lectin; Con A, Jack bean (Canavalia ensiformis) lectin; EcorL, West Indian coral tree (Erythrina corallodendron) lectin; Fuc, L-fucose; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; GlcNAc, N-acetyl-D- glucosamine; GS4, Griffonia simplicifolia (now Bandeirea simplicifolia) lectin IV; PHA-L, leucoagglutinating common bean (Phaseolus vulgaris) agglutinin; PNA, peanut (Arachis hypogaea) agglutinin; r.m.s.d., root-mean-square deviation; SBA, soybean (Glycine max) agglutinin; WBAI, basic winged bean (Psophocarpus tetragonolobus) agglutinin. Article No. jmbi.1998.2534 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 286, 1161±1177 0022-2836/99/091161±17 $30.00/0 # 1999 Academic Press