The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes

Abstract

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys65, Arg68, and Arg69, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.

Interplay between Membrane Curvature and Cholesterol: Role of Palmitoylated Caveolin-1

“…Protein lipidation is characteristic of many membraneassociated proteins that are involved in signal transduction (81). Atomistic and coarse-grain simulations have been used to complement experimental studies to analyze the conformational dynamics of lipidated membrane proteins (44,(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96). In particular, the effect of palmitoylation and farnesylation on the Ras family of proteins has been reported, such as their membrane localization (85), structural dynamics, and mode of binding to lipid bilayers (83,84,(86)(87)(88)(89).…”

“…In particular, the effect of palmitoylation and farnesylation on the Ras family of proteins has been reported, such as their membrane localization (85), structural dynamics, and mode of binding to lipid bilayers (83,84,(86)(87)(88)(89). Simulations with the Martini coarse-grain force field (39) have been successful in analyzing protein-lipid association as well as the organization and partitioning behavior of lipidated (particularly palmitoylated) proteins in bilayers (44,90,(92)(93)(94)(95). An important role of the lipid tail was suggested in anchoring the proteins to the membrane.…”

“…For the palmitoylated proteins, the palmitoyl tail was attached to the C-terminal cysteine residue (Cys133) using the Martini lipid parameters of the DPPC palmitoyl chain. The optimized protein-lipid parameters were obtained from a previous computational study on lipidated LC3 protein (44). Dihedral potentials were added to the backbone beads to maintain the secondary structure of the protein (30).…”

The conformational and mutational landscape of the ubiquitin-like marker for the autophagosome formation in cancer

“…Among the simulations present in the literature, each one focused on specific aspects of LC3B dynamics using all-atom molecular dynamics (MD) simulations with a single force field (Jatana et al, 2019;Liu et al, 2013;Nuta et al, 2018;L. Zhao et al, 2017) or coarse-grained approaches (Thukral et al, 2015).…”

Experimental and Computational Approaches to Study Membranes and Lipid–Protein Interactions

“…Similarly, despite a clear dependence on charged lipid types, a direct lipid interaction has not been observed in lipid-anchored proteins such as LC3. 104 Additional studies are required to delineate the role of specific and non-specific effects.…”