3.4. Gal-3 directly combined with TLR4 in vitro
To expound the relationship between Gal-3 and TLR4 in vitro, Gal-3 to be collocated with and bound to TLR4 was found by CO-IP assay, and the colocalization of Gal-3 and TLR4, which was examined by co-immunofluorescence staining, were increased after IS (Fig. 4A-B, C). These results suggested that Gal-3 might influence the effect of TLR4 in inflammation response and apoptosis in CNS diseases. To clarify the possible structural and functional interaction regions and sites between Gal-3 and TLR4, their binding effects were examined by computer-aided molecular docking, two proteins form a wide range of interactions, mainly including hydrogen bonding, salt bridging and π-π stacking. The salt bridge action is formed between the positively charged side chain of the alkaline amino acid Lys153 and the negatively charged carboxylic acid side chain group of the Acidic amino acid Glu198. The salt bridge action is the superposition of hydrogen bond action and charges action, belonging to a strong ion-type interaction. The π-π stacking effect is formed between the side chain benzene ring charge center of phenylalanine Phe498 (F498) on TLR4 and the phenol charge center of tyrosine Tyr132 (Y132) on the Gal-3 protein (Fig 4 D). These data indicated that Gal-3 and TLR4 exist combined in physical structure directly.