NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris.

The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14,500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains alpha and gamma, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using (1)H-(15)N HSQC spectra as a function of increasing concentrations of lactose, melibiose, D-galactose, methyl alpha-D-galactopyranoside and methyl beta-D-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain alpha had a slow or intermediate exchange regime on the chemical-shift timescale (K(d) = 10(-2) to 10(-1) mM), whereas that in subdomain gamma had a fast exchange regime for these sugars (K(d) = 2-6 mM). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.