STRUCTURAL FEATURES OF THE COMBINING SITE REGION OF ERYTHRINA-CORALLODENDRON LECTIN - ROLE OF TRYPTOPHAN-135

The role of Trp 135 and Tyr 108 in the combining site of Erythrina cor allodendron lectin (ECorL) was investigated by physicochemical charact erization of mutants obtained by site-directed mutagenesis, hemaggluti nation-inhibitin studies, and molecular modeling, including dynamics s imulations. The findings demonstrate that Trp 135 in ECorL: (1) is req uired for the tight binding of Ca2+ and Mn2+ to the lectin because mut ation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) co ntributes to the high affinity of methyl alpha-N-dansylgalactosaminide (Me alpha GalNDns) to the lectin; and (3) is solely responsible for t he fluorescence energy transfer between the aromatic residues of the l ectin and the dansyl group in the ECorL-Me alpha GalNDns complex. Dock ing of Me alpha GalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as requ ired for efficient fluorescence energy transfer, in one of the two pos sible conformations that this ligand assumes in the bound state. In th e W135A mutant, which still binds Me alpha GalNDns strongly, the dansy l group may partially insert itself into the place formerly occupied b y Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assu mes an open conformation. However, a small fraction of the W135A molec ules must be able to bind Me alpha GalNDns in order to explain the rel atively high affinity, as compared to galactose, still remaining for t his ligand. A model for the molecular events leading to inactivation o f the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed i n high-temperature dynamics simulations, appears not to be a required step.