Visualizing the Contact Activation System: A Cryo-EM Study of the Factor XII - C1-Esterase Inhibitor Complex

Sammanfattning

For decades, the blood plasma contact activation system has been recognized as a fundamental mediator of inflammation and thrombosis, with blood factor twelve (FXII) at its core. FXII and its associated partners in the blood plasma contact activation system are crucial for multiple biological processes which has made them attractive therapeutic targets. However, the lack of high-resolution 3D structures of FXII and associated macromolecules has hindered continued investigation. This project aims to address these gaps of knowledge by resolving 3D structures using cryo-EM. A robust protocol for expression and purification of FXII was developed using Pichia Pastoris as heterologous expression host. Followed by optimized purification using ÄKTA Flux S tangential flow filtration and size exclusion chromatography (SEC) to achieve high-yield production of highly purified FXII. However, chromogenic activity assays and nanoDSF thermal unfolding experiments suggest impaired activity and structural stability of the recombinant FXII when evaluated against commercial FXII. Likely due to the inability of Pichia Pastoris to replicate native-like post-translational modifications necessary for functional human FXII. Cryo-EM grid preparation, grid screening and data collection of the FXII:C1-INH complex was successful despite these issues. CryoSPARC data processing and evaluation produced a highly heterogenous dataset, which was used to generate a 3D reconstructed density map. Initial rigid-body fitting, suggest that the reconstructed density map does not represent a FXII: C1-INH complex. Rather, the density map revealed structural similarities to the FXII light chain protease domain connected to a truncated version of the heavy chain representing either domain pairs or individual domains. Further refinement of the reconstructed density map is required, but these findings may indicate a novel conformational state of FXII not yet seen in literature.