Assembly and Molecular Mechanism Studies on Membrane Remodeling Complex

Description

Membrane remodeling is critical for many aspects of cellular function, including the biogenesis of organelles and transport carriers, cell motility, and cytokinesis. Two of the best characterized protein families that participate in membrane remodeling are ADP-ribosylation factor (Arf) of small G protein and sorting nexin (SNX) family protein containing the Bin-Amphiphysin-Rvs (BAR) domain. Arfs and SNXs regulate membrane traffic and dynamics, such as modulating membrane lipid composition, defining tubular endosomal network, and organizing trafficking among endocytic and recycling pathways. Therefore, it is essential to understand how these proteins interact cell membrane. However, the molecular mechanism of membrane remodeling remains unclear, due to lack of three dimensional (3D) structure of the remodeling complex.This project aims to obtain the 3D structures of these remodeling complexes using cryo-EM experiment and molecular dynamics (MD) simulations. More specifically, the 3D structure of remodeling complexes includes two important interfaces, the protein-protein (PP) interface to stabilize the protein lattice structure, and the protein-membrane (PM) interface for the protein to bind to the membrane. Molecular protein lattice structure, interaction mechanisms, and key residues on the two interfaces will be investigated using experimental and computational methods.In order to reveal the PP interface, the mainland team will first obtain the electron density map of the complex structure of SNX1 (Arf6) protein on lipid tube using cryo-EM experiments. Atomic models will be fitted using UCSF Chimera. Afterwards, the PI will apply a computational method to further refine the protein lattice structure, and reveal key residues to maintain the lattice. Moreover, with respect to the PM interface, the PI will perform Monte Carlo calculations to search for the optimal binding orientation between protein and membrane; afterwards, all-atom MD simulations will be carried out to further refine the PM interface. Key residues responsible for the protein binding will be identified. In addition, the binding specificity of SNX1 to lipid molecules will be investigated from the energy point of view. To verify the simulation predictions, the mainland team will carry out in vivo and in vitro mutagenesis experiments, and quantify the protein binding rate as well as the tubulation rate.The computational and experimental results will provide new insights into the molecular mechanism of membrane remodeling by SNXs and Arfs proteins, elucidate how proteins assemble on the membrane surface, and make it possible to further investigate their functional mechanism in intracellular transport pathway in the future.