The ligand-binding loops in the tunicate C-type lectin TC14 are rigid

C-Type lectin-like domains are very common components of extracellular prot eins in animals. They bind to a variety of ligands, including carbohydrates , proteins, ice, and CaCO3 crystals. Their structure is characterized by lo ng surface loops in the area of the protein usually involved in ligand bind ing. The C-type lectin TC14 from Polyandrocarpa misakiensis specifically bi nds to D-galactose by coordination of the sugar to a bound calcium atom. We have studied the dynamic properties of TC14 by measuring N-15 longitudanal and tranverse relaxation rates as well as {H-1-N-15} heteronuclear NOEs. R elaxation rates and heteronuclear NOE data for holo-TC14 show minimal varia tions, indicating that there is no substantial difference in rigidity betwe en the elements of regular secondary structure and the extended surface loo ps. Anisotropic tumbling of the elongated TC14 dimer can account for the ma in fluctuations in relaxation rates. Loss of the bound calcium does not sig nificantly alter the internal dynamics, suggesting that the stability of th e loop region is intrinsic and not dependent on the coordination of the cal cium ion. Chemical shift differences between the holo and apo form show tha t main structural changes occur in the calcium-binding site, but smaller st ructural changes are propagated throughout the molecule without affecting t he overall fold. The disappearance of two resonances for residues following the conserved cis-proline 87 (which is located in the calcium-binding site ) in the apo form indicates conformational change on an NMR time scale betw een the cis and trans configurations of this peptide bond in the absence of calcium. Possible implications of these findings for the ligand binding in C-type lectin-like domains are discussed.