proteins STRUCTURE O FUNCTION O BIOINFORMATICS STRUCTURE NOTE Crystal structure of the putative carbohydrate recognition domain of human galectin-related protein Martin Andreas Wa ¨lti, 1y Ste ´phane Thore, 2y Markus Aebi, 1 and Markus Ku ¨nzler 1 * 1 Institute of Microbiology, ETH Zu ¨rich, Wolfgang-Pauli-Str. 10, CH-8093 Zu ¨rich, Switzerland 2 Institute of Molecular Biology and Biophysics, ETH Zu ¨rich, Schafmattstr. 20, CH-8093 Zu ¨rich, Switzerland Key words: HSPC159; LOC29094; X-ray crystallography; sugar-binding; legume lectin-like. INTRODUCTION Galectins are b-galactoside binding proteins sharing a conserved carbohydrate recognition domain (CRD). 1 Galectin-encoding genes are present in the genomes of a wide variety of eukaryotic organisms ranging from ma- rine sponges up to humans. 2 They are however restricted to the animal kingdom and to the subclass of agaricomy- cetidae in the fungal kingdom. Members of the galectin family can be assigned to one of three subgroups: The prototype subgroup having only one CRD per monomer, the tandem-repeat type having two CRDs and the chi- mera type which possesses a long peptide extension in addition to its CRD. 3 The highly conserved residues responsible for b-galac- toside association have been defined based on the X-ray structures of galectin family members or respective CRDs in complex with their carbohydrate ligands. 4–9 Residues His49, Asn51, Arg53, Asn62, and Glu72 recognize the b-galactoside ligand via specific hydrogen bonds, while Trp69 confers binding affinity through hydrophobic stacking with the galactose moiety [numbering of human galectin-7 (hGal-7)]. However, within the galectin family, there are a growing number of proteins containing a galectin CRD based on sequence similarity, but which lack some of the strictly conserved amino acids responsi- ble for b-galactoside binding and accordingly do not dis- play significant affinity for b-galactosides. Members of this so-called galectin-related protein (GRP) family are galectin-related interfiber protein (GRIFIN), the man- nose-binding Charcot-Leyden crystal protein, Coprinopsis cinerea GRP (CGL3) and human GRP, to name a few. Human GRP contains a short N-terminal segment of unknown function and a putative CRD, which shares consensus amino acids at 51 out of the 64 most highly conserved residues in other galectins 2 (see Fig. 1). No ligand has so far been identified. To get insight into the function of this GRP highly conserved among animals, we have determined the three-dimensional structure of the GRP CRD fragment at 2.0 A ˚ resolution. METHODS Cloning, expression, and purification The HSPC159 gene, coding for human GRP, was amplified by PCR using cDNA generated from mRNA of HEK293 cells as a template. The PCR product was cloned into pBAD-TOPO (Invitrogen), which served as a tem- plate for the generation of a plasmid for bacterial expres- sion of the N-terminally truncated and his-tagged GRP (GRP-C; residues 38–172, accession NP_054900). The site of truncation was chosen according to a GRP expression and crystallization study. 13 The respective insert was gen- erated by PCR using the forward oligonucleotide primer NdeI-His8-GRP (5 0 -CAT ATG GGC CAT CAT CAT CAT CAT CAT CAT CAC AGC GGC GTT CCA TTT TGT *Correspondence to: Markus Ku ¨nzler, Institute of Microbiology, ETH Zu ¨rich, Wolfgang-Pauli Str. 10, CH-8093 Zu ¨rich, Switzerland. E-mail: markus.kuenzler@micro.biol.ethz.ch y Martin Andreas Wa ¨lti and Ste ´phane Thore contributed equally to the work. Received 31 January 2008; Revised 7 March 2008; Accepted 14 March 2008 Published online 23 April 2008 in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/prot.22078 804 PROTEINS V V C 2008 WILEY-LISS, INC.