The Structure of a Tunicate C-type Lectin from Polyandrocarpa misakiensis Complexed with D-Galactose Se  bastien F. Poget 1 *, Glen B. Legge 1 , Mark R. Proctor 1 , P. Jonathan G. Butler 2 , Mark Bycroft 1 and Roger L. Williams 2 1 Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lens®eld Road Cambridge, CB2 1EW, UK 2 Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 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 mM, and con®rmed the speci®city of TC14 for D-galactose and related monosaccharides. We have determined the 2.2 A Ê X-ray crystal structure of Polyandrocarpa lectin complexed with D-galactose. Analytical ultracentrifu- gation revealed that TC14 behaves as a dimer in solution. This is re¯ected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal b-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 a-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 monosac- charide speci®city 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 speci®city. These elements are, however, arranged in a very different way than in an engineered galactose-speci®c mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions. # 1999 Academic Press Keywords: invertebrate C-type lectin; carbohydrate recognition; galactose speci®city; calcium-binding af®nity; dimeric interface *Corresponding author Introduction Molecular recognition in biological systems is often based on interactions between oligosacchar- ide structures and corresponding receptor proteins. The C-type lectins are a family of extracellular carbohydrate recognition proteins characterised by a common sequence motif of 115 to 130 amino acid residues. This domain, called the carbohydrate rec- ognition domain (CRD), usually shows speci®c, but weak (with dissociation constants in the mM range) calcium-dependent binding to a variety of monosaccharides (Drickamer, 1993). C-type lectin CRDs are found as building blocks in a variety of Present address: G. B. Legge, Department of Molecular Biology MB-2, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Abbreviations used: CRD, carbohydrate recognition domain; ESEL, human E-selectin; HLIT, human lithostathine; HTNT, human tetranectin; ITC, isothermal titration microcalorimetry; MBPA, rat serum mannose- binding protein; MBPC, rat liver mannose-binding protein; TC14, Polyandrocarpa lectin (tunicate-derived C- type lectin of 14 kDa); 6-GPGP, 6-b-D-galactopyranosyl- D-galactopyranose; 4-GPGP, 4-a-D-galactopyranosyl-D- galactopyranose; DTT, dithiothreitol. E-mail address of the corresponding author: sfp22@cam.ac.uk Article No. jmbi.1999.2910 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 290, 867±879 0022-2836/99/290867±13 $30.00/0 # 1999 Academic Press