The three-dimensional crystal structure of carp fish egg lectin

A novel lectin present in carp eggs was recently discovered and characterized in our laboratory and named Fish Egg Lectin (FEL). FEL is a glycoprotein of 238 amino acids with a molecular weight of 26.6 kD. The sequence analysis showed a significant similarity with a lectin present in the plasma and haemocytes of the horseshoe crab Tachypleus tridentatus, Tachylectin 1, involved in the non specific immune response of the invertebrate. Biochemical studies performed by the research group of Prof. Galliano (University of Pavia) have evidenced that this lectin is present in solution as a monomer and can bind to saccharides on the surfaces of Gram positive and negative bacteria. N- acetylglucosamine and N-acetylgalactosamine show the highest affinity among the monosaccharides while the lectin has little or no affinity for simple sugars or disaccharides as lactose. Immunohistochemical studies demonstrated that FEL is an egg specific protein and that it is absent from every other tissue in fish. It is the first time that a lectin like this is described in a vertebrate and its real biological role has still to be understood. This thesis focuses on the three-dimensional structure study of FEL by X- ray diffraction analysis of single crystals. After numerous trials, diffraction quality crystals of the native protein were prepared using a solution of 15% 2-propanol, 20% PEG 6000, 0.1 M HEPES pH 7.5 as precipitant. They are orthorhombic and belong to the space group P212121, with cell parameters a = 44.72 Å, b = 72.28 Å and c = 167.59 Å. The phase problem was solved by the multiple isomorphous replacement method. Several derivatives were prepared by soaking protein crystals in mother liquor containing the heavy atom compound and the diffraction data were collected and analysed. The best three derivatives (K2PtCl4, Hg(Ac)2 and UO2(Ac)2) were used in the phase determination process. 2.5 Å resolution electron density maps were calculated and the molecular model was built and refined. In the asymmetric unit this lectin is present as a homodimer in which both monomers have a six β-propeller fold, a highly symmetric structure made up six “blades” of antiparallel β-strands that surround a central cavity. The analysis of intermolecular contacts and the change in accessible surface area upon dimer formation indicate that the dimeric form of FEL is the real physiological unit and not an artefact of crystal packing. A calcium-binding site was identified and described in the central tunnel of each monomer. The binding of monosaccharides was investigated by soaking FEL crystals in mother liquor containing 0.1 M N-acetylglucosamine and collecting the diffraction data of the complex. Only one GlcNAc binding site was identified in one of the monomers, in contrast with the presence of multiple binding sites described for other β-propeller lectins. The evidence of a single site, especially in a homodimer, is more likely due to the method used for complex preparation or to the low affinity for the ligand rather than to a real peculiarity of this protein. The binding of GlcNAc resembles that described for another lectin of Tachypleus tridentatus, Tachylectin 5A, with the acetyl group located in a hydrophobic cavity of the molecular surface and the glycosidic ring exposed to the solvent. Two other crystal forms were obtained and solved by molecular replacement with the coordinates of a monomer as search probe. A second orthorhombic form grows more slowly in the same conditions of the first one and it can be considered the result of a rearrangement of the crystal packing: the asymmetric unit is basically identical in the two forms and the unit cell differs only for the shorter length of the c axis. The third crystal form is trigonal and belongs to the space group P3. It was obtained under different crystallization conditions and the asymmetric unit is a hexamer arranged as a trimer of dimers. The analysis of the intermolecular contacts and the orientation of the monomers in each dimer revealed that they are very similar to each other and to those that make up the asymmetric unit of the orthorhombic forms. The presence of the same structural unit in three different crystal forms reinforces the assumption of the physiological dimeric nature of this lectin.