A programmable lipid-triggered allosteric site modulates LC3 LIR receptor binding activity
Membrane recruitment is a fundamental regulator of protein function. However, the allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. In autophagy, the ubiquitin-like protein LC3 is lipid-anchored to autophagosomes, where it is essential for receptor recruitment and vesicle formation. While LC3-receptor interactions are structurally well defined, how membrane engagement governs LC3 functional dynamics has remained enigmatic. Here, we uncover that membrane binding triggers a major conformational transition in LC3, exposing functional pockets that are occluded in its cytosolic form. We demonstrate that this shift is mediated by dynamic coupling between the allosteric site (α3-loop5-β3-loop6) and the functional binding pockets. To conclusively test this mechanism, we utilised an ensemble-based protein design strategy guided by molecular dynamics to engineer the allosteric site. From a series of mutants, two variants emerged that stabilized LC3 conformation in either active or inactive state on the membrane. X-ray crystal structures of mutant LC3, biophysical assays, super-resolution microscopy, and TEM confirmed that the activated allosteric site mutant facilitates receptor binding and cargo capture. In contrast, the inactive variant is functionally inert on the membrane. Our work identifies a fundamental lipid-triggered allosteric site in LC3 that is critical for autophagy regulation and broader implications of membrane-dependent reprogrammable protein activities. The allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. Here, the authors identify a lipid-triggered allosteric site in LC3 that remodels its receptor-binding interface upon membrane interaction. This site can be engineered to precisely tune LC3-LIR interactions, offering a new strategy to regulate selective autophagy.