Figure 4
Interaction/binding of rLsa21 with TLR2 and TLR4.
(A) HEK293-TLR2, HEK293-TLR4 and HEK293-vector cells were cultured in cell imaging dishes and then incubated with rLsa21 (2 μg/ml) or rLsa21 (2 μg/ml) treated with Proteinase K for 30 min. After washing, the cells were fixed with 4% PFA and then sequentially incubated with mouse anti-rLsa21 serum and FITC Goat anti-mouse IgG. After washing, mounting medium was added and then viewed and photographed with confocal fluorescence microscopy. Data are representative of three independent experiments. (B) TLR2−/−, TLR4−/− or TLR2−/−/TLR4−/− DKO macrophages cell lines were incubated with rLsa21 (2 μg/ml) or rLsa21 (2 μg/ml) treated with Proteinase K for 30 min. After washing cells were fixed and stained with respective antibodies as described in materials and methods. (C) Molecular modeling of interaction between Lsa21 (green) and LRR domain of TLR2 (blue). All 10 top ranking docking complexes showed that the Lsa21 binding site lies at convex region of TLR2 and its binding region overlaps with ligand binding and TLR1-TLR2 hetro-dimerization region (LRR- 10, 11, 12). Molecular modeling of interaction between Lsa21 (green cartoon) and TLR4 (blue cartoon). Out of 10 top ranking docking complexes, in three docking complexes Lsa21 was observed to bind to concave region and interact with C-terminal and remaining seven complexes it docked on the N-terminal region of TLR4. BF- indicates bright field, PK indicates Proteinase K treatment.
