Structure of the Lectin IV of Griffonia simplicifolia and its Complex with the Lewis b Human Blood Group Determinant at 2·0 Å Resolution

Abstract

The structures of the fourth lectin isolated from Griffonia simplicifolia (GS4) and its complex with the methyl-glycoside of the Lewis b human blood group determinant (Le b-OMe) are reported at high resolution. The native GS4 crystal is isomorphous with the complexed GS4 crystal. The space group is P4 22 12 with unit cell dimension a = 78·9 Å, c = 89·1 Å with one subunit of the lectin (bound to 1 Le b-OMe in the complex) in the crystallographic asymmetric unit. The native GS4 structure was solved by the molecular replacement technique and least-squares refined (PROLSQ and X-PLOR). The orientation of the Le b-OMe tetrasaccharide in the complex was established from a 2·8 Å difference map with coefficients ( F complex- F native) and calculated phase angles from the native model. Both the final native and complex GS4 models consist of 1904 protein non-hydrogen atoms, one sulfate ion, one Ca ion, one Mn ion and three covalently-bound sugar residues N-linked to Asn 18. In addition, the complex model has 47 Le b-OMe non-hydrogen atoms. The two structures have 135 water molecules in common in addition to eight and nine unique water molecules in the native and complex structures, respectively. The root-mean-square deviations from ideal bond distances and angles are 0·016 Å, 3·2° and 0·016 Å, 3·0°, for the native and complexed GSA, respectively. The R index for all unique data from 8 to 2·0 Å is 0·187 for the native (19,204 reflections) and 0·181 for the complex (19,212 reflections). The tertiary structure of each subunit is similar to that of other leguminous lectins but the quaternary structure of the molecular dimer is different from that of any other lectin reported to date. The co-ordination about the Ca ion is pentagonal bipyramidal (with 1 long Ca 2+-oxygen bond) and the co-ordination about the Mn ion is octahedral. Two conserved residues (Asp149 and Ser155) appear to be important because they are hydrogen-bonded to each other and to groups that co-ordinate the Mn ion. There are three cis-peptides in the polypeptide chain; two involve non-proline residues, one of which is homologous with other leguminous lectins and the other is unique to GS4. The two non-proline cis-peptides are located in the carbohydrate-binding site and are important for the specificity of the lectin. The molecular recognition of Le b-OMe by GS4 involves both polar and extensive non-polar interactions. Some key polar interactions of GS4 involve the side-chains of Asp89 and Asn135 and the peptide NH of Gly107, which are near the center of the shallow depression which forms the carbohydrate-binding site. Several non-polar protein-carbohydrate interactions include six aromatic amino acid residues of GS4, namely Try105, Phe108, His114, Trp133, Trp 138 and Tyr223 which make up a large portion of the carbohydrate-binding site. Several amino acid residues in the carbohydrate-binding site adjust positions by approximately 1 to 2 Å in order to optimize the hydrophilic and hydrophobic interactions in the complex; these rearrangements are similar in magnitude to those found to occur in antibody-antigen complexes. These results suggest that common aspects of receptor sites in proteins may be the occurrence of a significant number of aromatic residues and adjustments of <2 Å in residues upon formation of a complex.