The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.
C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.
Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. sfp22@cam.ac.uk
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D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D005690:Galactose / Q000737:chemistry*; D037102:Lectins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D013997:Time Factors; D014461:Ultracentrifugation; D014561:Urochordata / Q000737:chemistry*
D037102:Lectins; D011994:Recombinant Proteins; D002118:Calcium; D005690:Galactose