Structural characterization of galectin-3 and galectin-related protein by x-ray crystallography

Laburpena

Galectins are a family of beta-galactoside-binding proteins located in a wide range of organisms, from mammals to fungi and prokaryotes, where they exert functions that can be mediated by both carbohydrate-protein and protein-protein interac-tions. Galectins were classified based on their structural features in prototype, tandem-repeat and chimeric type. Prototype galectins contain one CRD per subu-nit and can form homodimers whereas tandem-repeat galectins are heterodimers of two non-identical CRDs linked by a peptide. The chimeric galectins have a distinctive N-terminal domain comprised of a lead peptide and nine poly-Pro/Gly repeats (namely, N-PG) linked to the C-terminal CRD. In humans, the only chi-meric type is Galectin-3 (Gal-3). The N-PG module had eluded structural resolu-tion so far, albeit the many reports on the CRD structure. Using a protein engi-neering approach, Gal-3 constructs with different lengths of the N-PG were pro-duced and set to crystallize. A particular construct, with repeats VII to IX and the lead domain (Gal-3[N VII-IX]) crystallized with high-reproducibility. The crystals belonged to the orthorhombic space group P212121 and had a resolution of 2.2 Å. Further studies were performed in this construct and the full-length protein by SAXS. Taken together, the results on the structural characterization of Gal-3[N VII-IX] allow to ascertain the position of key amino acids in the N-terminal that play fundamental roles in Gal-3 function, most importantly the position of an apoptosis- and metastasis-related phosphorylation site, Ser6. This new structure with the N-terminal section sets the path for the elucidation of full-length Gal-3 structure and its pharmacological applications. To date, a total of 15 members have been identified in the galectin family plus a few related proteins that closely share their sequence. A comprehensive genetic mapping of this family identified one particular Galectin-Related Protein (former-ly known as HSPC159, now GRP) located in bone marrow that has a high degree of identity with galectins sequences, sharing their jelly-roll characteristic topology. This GRP is present only in vertebrates, unlike its ubiquitous relatives. Interesting-ly, GRP does not bind -galactosides. Human GRP structure has been reported twice with no further information on its function. In order to study in detail the entirety of the galectin family for identifying themes of divergence and recogni-tion within a limited number of proteins, Gallus gallus GRP was chosen as model. Crystals of this protein belonged to the body-centered orthorhombic space group I212121 and had a resolution of 1.5 Å. Their crystallographic resolution showed the particular hallmarks that hinder beta-galactoside binding. This structure and its properties were further studied by SAXS and AUC.