Active Components of Leptospira Outer Membrane Protein LipL32 to Toll-Like Receptor 2

Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that beta 1 beta 2, loop-alpha 3-loop, and alpha 4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32 Delta N beta 1 beta 2 significantly decreased the affinity to TLR2 while LipL32 Delta C alpha 4 slightly reduced it. Interestingly, LipL32 Delta Cen alpha 3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cen alpha 3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through N beta 1 beta 2 and C alpha 4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.