The Structural Basis Of Caspase-11 Inflammasome Signal Activation

Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS.LPS is an important component of gram-negative bacterial cell wall.Under physiological conditions,gram-negative bacteria produce outer membrane vesicles(OMVs),which carrying LPS enters the cytocol through endocytosis of host cells.GBPs(guanylate-binding proteins)helps releasing LPS from OMVs,which allows caspase-11 recognize LPS and leads to GSDMD cleavage.Caspase-11 contains two components,an N-terminal caspase recruitment domain(CARD)and a C-terminal catalytic domain.The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11.However,the activation mechanism of caspase-11 remains unclear.CARD subfamily members are formed as six α-helices,and which also belong to DD superfamily.Caspase-11 CARD consists of around 59 amino acids and it can transmit signals through homotypic oligomerization and then results in activation of caspase-11.To date,the crystal structure of caspase-11 catalytic domain and GSDMD protein complex has been solved,and catalytic domain has the highly conservative structure of the caspase family.However,there is no crystal structure of caspase-11 CARD been solved already.And how does caspase-11 been activated still remain unknown.There are three main results in this thesis:(1)In this study,we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution.And we solved the crystal structure of caspase-11 CARD with an N terminal MBP.In an asymmetric unit,there are four homologous molecules.Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1-2 helix mediating homotypic CARD interaction.The structure of caspase-11 CARD did share a conserved six-helical bundle fold formation of CARD subfamily members.The conformation change of H1,H2 helix provided a hydrophobic interface of CARD which was important for the oligomerization of caspase-11.(2)Mutations of the helix Hl-2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells.And LPS was unnecessary for caspase-11 activation.(3)We found that both CARD and a flexible loop region connecting CARD to the catalytic domain are essential for caspase-11 aggregation,which was confirmed by truncations of CARD.Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model.This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition,leading to the caspase-11 activation.