Membrane lipids switch LC3 into its active form: researchers identified and reprogrammed an allosteric control site in autophagy
Lipids in the autophagosome membrane, the membrane-bound compartment used for cellular disposal, change the conformation of LC3 and help it capture cargo
On August 28, researchers reported in an article in Nature Communications that anchoring LC3 to the autophagosome membrane changes its conformation and promotes binding to cellular cargo receptors. Computational analysis identified a distant regulatory site, and the researchers then tested its role using two modified LC3 variants.
Autophagy is a cellular disposal process. The cell packages damaged proteins and worn cellular components into a double-membrane vesicle called an autophagosome. A lysosome, a cellular compartment containing degradative enzymes, then destroys the contents of the autophagosome. LC3 is anchored to the autophagosome membrane and binds cargo receptors, which act as intermediary proteins. One such receptor, p62, connects LC3 to material that the cell needs to remove.
The lipid anchor has long explained how LC3 becomes attached to the membrane. What remained uncertain was how this attachment affects receptor binding. The authors used molecular dynamics, a computational method that models atomic motion, to compare free LC3 with LC3 attached to a model membrane. In the simulations, lipids reorganized internal contacts within LC3 and made its receptor-binding sites more accessible.
The calculations identified a distant region of the protein that briefly contacts lipids. Motion in this region was correlated with motion in the receptor-binding sites of LC3, located about one and a half nanometers away. This produced a testable hypothesis: stabilizing the region should strengthen receptor binding, whereas increasing its mobility should weaken binding.
Of 13 designed LC3 variants, the researchers selected two variants containing triple amino acid substitutions with opposite predicted effects. In an experiment using purified protein, the active variant bound a short p62 fragment almost twice as strongly as unmodified LC3, whereas the inactive variant bound it about one and a half times more weakly. X-ray structures showing the atomic arrangement of the active variant confirmed the predicted protein rearrangement both before p62 binding and in the complex with p62.
In nutrient-deprived mouse liver cells, the active variant bound p62 about eight times more strongly than unmodified LC3. The proteins were also found near each other more often in a stably maintained cell line. The authors separately confirmed that both substitutions preserved the usual LC3 conformation and its membrane contacts. Removing the lipid anchor from the protein sharply reduced p62 binding, showing that membrane attachment remains part of the mechanism.
Through LC3, membrane lipids make the sites used by the autophagosome to bind receptors and their associated cargo more accessible. The study provides an experimental method for examining this regulator of cellular disposal independently from other components of the process.