biostudies-literatureYu JNational Institutes of HealthHuck Institutes of the Life SciencesNational Institute of General Medical Sciences110543https://www.ebi.ac.uk/biostudies/studies/S-EPMC12446773biostudies-literatureUnknown301(9)Picornavirus infection leads to the production of new membranous structures that are enriched in phosphoinositide lipids. The multifunctional 3C protease interacts with these phosphoinositide-enriched membranes. We performed paramagnetic relaxation enhancement NMR experiments to gain insight into the molecular determinants driving these interactions. We found that 3C interacts with lipid membranes through both its positively charged N-terminal helix and the 80 to 90's region, previously proposed to interact with RNA. Experiments using different pH and salt concentrations suggest that the interaction is primarily electrostatically driven. The optimized lipid membrane systems developed here provides molecular insights into the 3C-membrane interactions and can be leveraged to study peripheralThe Journal of biological chemistryMolecular determinants of picornavirus 3C protease binding to phosphoinositide-enriched lipid membranes.PMC12446773AI104878P41GM111135S10 OD030490Winston DSBoehr DDYu JfalseMolecular determinants of picornavirus 3C protease binding to phosphoinositide-enriched lipid membranes.Picornavirus infection leads to the production of new membranous structures that are enriched in phosphoinositide lipids. The multifunctional 3C protease interacts with these phosphoinositide-enriched membranes. We performed paramagnetic relaxation enhancement NMR experiments to gain insight into the molecular determinants driving these interactions. We found that 3C interacts with lipid membranes through both its positively charged N-terminal helix and the 80 to 90's region, previously proposed to interact with RNA. Experiments using different pH and salt concentrations suggest that the interaction is primarily electrostatically driven. The optimized lipid membrane systems developed here provides molecular insights into the 3C-membrane interactions and can be leveraged to study peripheral2025-01-01T00:00:00Z2025 Jul2026-06-03T14:32:15.214Z2026-04-28T03:11:09.904ZS-EPMC124467734074983310.1016/j.jbc.2025.110543