Natural triterpenoid-aided identification of the druggable interface of HMGB1 occupied by TLR4

HMGB1 interacts with TLR4 to activate the inflammatory cascade response, contributing to the pathogenesis of endogenous tissue damage and infection. The immense importance of HMGB1-TLR4 interaction in the immune system has made its binding interface an area of significant interest. To map the binding interface of HMGB1 occupied by TLR4, triterpenoids that disrupt the HMGB1-TLR4 interaction and interfere with HMGB1-induced inflammation were developed. Using the unique triterpenoid PT-22 as a probe along with photoaffinity labeling and site-directed mutagenesis, we found that the binding interface of HMGB1 was responsible for the recognition of TLR4 located on the "L" shaped B-box with K114 as a crucial hot-spot residue. Amazingly, this highly conserved interaction surface overlapped with the antigen-recognition epitope of an anti-HMGB1 antibody. Our findings propose a novel strategy for better understanding the druggable interface of HMGB1 that interacts with TLR4 and provide insights for the rational design of HMGB1-TLR4 PPI inhibitors to fine tune immune responses.