The crystal structure of the α5β1 integrin reveals conformational changes and amino acids important for ligand binding.

Podoplanin is a transmembrane O-glycoprotein that binds to C-type lectin-like receptor 2 (CLEC-2). The O-glycan-dependent interaction seems to play crucial roles in various biological processes, such as platelet aggregation. Rhodocytin, a snake venom, also binds to CLEC-2 and aggregates platelets in a glycan-independent manner. To elucidate the structural basis of the glycan-dependent and independent interactions, we performed comparative crystallographic studies of podoplanin and rhodocytin in complex with CLEC-2. Both podoplanin and rhodocytin bind to the noncanonical "side" face of CLEC-2. There is a common interaction mode between consecutive acidic residues on the ligands and the same arginine residues on CLEC-2. Other interactions are ligand-specific. Carboxyl groups from the sialic acid residue on podoplanin and from the C terminus of the rhodocytin α subunit interact differently at this "second" binding site on CLEC-2. The unique and versatile binding modes open a way to understand the functional consequences of CLEC-2-ligand interactions.

1,2-␣-L-Fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fuc␣1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 Å resolution. The AfcA Fuc domain is composed of four regions, an N-terminal ␤ region, a helical linker, an (␣/␣) 6 helical barrel domain, and a C-terminal ␤ region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu 566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn 423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2-fucosyllactose, and its location is nearly identical near the O1 atom of ␤-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.