Expression and Isolation of Full-Length Human TLR4 in Nanolipoprotein Particles for Future Structural Analysis via Cryo-EM 4289

Abstract Description The innate immune system is the first line of defense against pathogens, responding rapidly through the strategic deployment of immune cells at common infection sites, such as the skin and airways. These immune cell sentinels express various highly conserved receptors, including Toll-like receptors (TLRs), which recognize and bind to pathogen components, triggering a signaling cascade that initiates an immune response. Despite their importance, a complete structural characterization of full-length TLRs, including their intact extracellular domain (ECD), transmembrane domain (TMD), and Toll/interleukin-1 receptor (TIR) domain organization, has not yet been reported. In this study, we explored the expression and isolation of human TLR4 incorporated in nanolipoprotein particles (NLPs) using cell-free systems from E. coli, CHO cells, and rabbit endotoxin-free reactions, alongside a cell-based transfection method with Expi293F cells derived from the HEK293 cell line. We characterized the isolated NLP-bound human TLR4 proteins and assessed their functionality in the presence and absence of the TLR4 activating ligand, lipopolysaccharide (LPS), using a TLR4 knockout monocyte cell line (THP1 Dual MD2-CD14-KO-TLR4). Our findings indicate that the cell-based NLP-bound human TLR4 yielded functional proteins suitable for time-resolved single-particle cryo-electron microscopy (cryo-EM), paving the way for elucidating the full-length TLR4 structure with intact ECD-TMD-TIR organization. Funding Sources This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore. National Laboratory under Contract DE-AC52-07NA27344. Support was provided by LDRD grant 50888. LLNL-ABS-2001103 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)