Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB

• DOI: 10.1016/j.cell.2024.03.022
• Publication Date: 2024-04-25T14:37:17Z
• Authors: Benjamin L. Lampson; Ana S. Ramίrez; Marta Baro; Lixia He; Mudra Hegde; Vidyasagar Koduri; Jamie L. Pfaff; Ruth E. Hanna; Julia Kowal; Nitin H. Shirole; Yanfeng He; John G. Doench; Joseph N. Contessa; Kaspar P. Locher; William G. Kaelin
• Keywords: Lipopolysaccharides; Inflammation; Glycosylation; Cryoelectron Microscopy; NF-kappa B; Membrane Proteins; Toll-Like Receptor 4; Mice; HEK293 Cells; Hexosyltransferases; Catalytic Domain; Humans; Animals; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; Signal Transduction

ISSN:0092-8674<br/>Cell, 187 (9)<br/>Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.<br/>ISSN:1097-4172<br/>