PR003052 (Project)

Description:Lipid-binding domains, traditionally isolated from natural proteins, are essential tools for probing membrane lipid dynamics and specialized cellular compartments. Despite diverse applications, a general strategy for their engineering remains elusive. Here, we present a robust and high-throughput method for monitoring protein-lipid interactions, named the Cell surface Liposome Binding (CLiB) assay. Employing the assay, we conducted directed evolution of the PX domain from SnxA, isolating high-affinity variants specific for phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. Combining the CLiB assay with next-generation sequencing enabled parallel analysis of >6,000 clones, comprehensively identifying key residues critical for lipid binding. An engineered variant, PX-SnxAGV, functioned as a lipid biosensor in yeast and mammalian cells, visualizing PI(3,5)P2-enriched membrane subdomains upon hyperosmotic shock and during microautophagy, thereby suggesting localized PI(3,5)P2 synthesis within spatially restricted regions. This study opens a new avenue for on-demand generation of lipid-binding probes, facilitating the discovery of membrane compartments characterized by unique lipid compositions.