The C-terminal domain (Ch; C-half) of the R-type earthworm 29-kDa lectin (EW29), isolated from the earthworm Lumbricus terrestris, has two sugar-binding sites, in subdomains α and γ, and the protein uses the two sugar-binding sites for its function as a single domain-type haemagglutinin. Our previous NMR titration experiments showed that the α sugar-binding site is a high-affinity site and the γ sugar-binding site is a low-affinity site. However, it remains unclear why the α sugar-binding site of EW29Ch binds to lactose much more strongly because the crystal structure of lactose-bound EW29Ch showed that the interaction between the α sugar-binding site and lactose was almost same as that between the γ sugar-binding site and lactose. In the present study, we have determined the NMR structure of EW29Ch in the sugar-free state and performed 15N relaxation experiments for EW29Ch in both the sugar-free state and the lactose-bound states. The conformation of EW29Ch in the sugar-free state was similar to that of EW29Ch in complex with lactose. Conformational changes upon binding of lactose were observed only for the α sugar-binding site. By contrast, the 15N relaxation experiments revealed a conformational exchange at the α sugar-binding site in the sugar-free state, which was suppressed in the lactose-bound state. The conformational exchange phenomenon observed for the α sugar-binding site was not observed for the γ sugar-binding site. Differences in the conformational change and the backbone dynamics between subdomains α and γ may be associated with the difference of the sugar-binding modes between the two sugar-binding sites.
Structural data for the NMR structure of EW29Ch in the sugar-free state have been deposited in the Protein Data Bank database under accession number 2RST.