Mutational studies of the Gal/GalNAc-specific leguminous lectin from the seeds of Erythrina corallodendron (ECorL), based on the 3-D structure of the ECorL-lactose complex [Shaanan et al., (1991) Science 254, 862], have shown that three combining site residues, Asp 89, Asn 133 and Phe 131 are essential for ligand binding by the lectin. They have also led to the proposal that a similar constellation of amino acids functions likewise in other legume lectins, irrespective of their specificity (except that Phe can be replaced by other aromatic residues). [Adar & Sharon (1996) Eur. J. Biochem., 239/3, 668-674].
ECorL binds MealphaN-dansylgalactosaminide (MeGalNDns) (Kass 3.5 x 10^5) much stronger than Gal/GalNAc (Kass 1-2 x 10^3). Modeling by manual docking demonstrates that the dansyl group of the ligand fits snugly into a cavity surrounded by Tyr108, Pro134 and Trp135 in the combining site of the lectin. Mutants Y108A and Y108T have the same specificity as ECorL and the same affinity for MeGalNDns showing that Tyr 108 does not play a role in binding of the dansyl. Pro134 is unlikely to contribute to ligand binding, since it occupies a variable position in the primary sequence of the lectin. Mutants W135Y and W135A still bind MeGalNDns strongly (ca. 100 times more than galactose by ECorL). Spectrofluorimetric studies establish that Trp135 is responsible for all the energy transfer between the tryptophans of ECorL and the dansyl of MeGalNDns, since no such transfer is observed with W135A. An unusual property of W135A is that it loses readily the bound Mn2+ and Ca2+ upon dialysis under conditions which they are retained by the native lectin and its many mutants, including W135Y, suggesting that in W135A, the metal ions are weakly held.
Molecular dynamics (MD) simulations show that the indole of Trp135 is parallel to the dansyl allowing efficient energy transfer between these two aromatic moieties. Comparison with other legume lectins reveals that the position equivalent to 135 of ECorL, which is part of the metal binding site, is always occupied by Trp, Tyr, Phe or Ile that interacts with the side chain of the hydrophobic residue (Ile or Val) at position 150, thus fixing the geometry of the metal binding site. The loss of this important conformational support upon replacement of Trp by Ala makes the geometry of the metal-binding site less stable, as demonstrated by MD simulations, and exposes the metals to the solvent, thus weakening their binding by the lectin.