Molecular mechanism of GTP binding- anddimerization-induced enhancement ofSar1-mediated membrane remodeling

The Sar1 GTPase initiates Coat Protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the Endoplasmic Reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in which GTP binding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino-terminus were not resolved in crystal structures, and experimental studies have suggested that the amino-terminus of Sar1 is solvent-exposed in the absence of membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membrane binding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1-23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membrane binding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10-20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane binding and remodeling activities in a concentration dependent manner, paving the way toward a better understanding COPII-mediated membrane transport. Significance StatementAmphipathic helices play established roles in penetrating lipid bilayers to produce local membrane curvature. The degree of curvature generated has been suggested to depend on the penetration depth of the amphipathic helix, its amino acid sequence, and the protein volume inserted into the membrane. However, the relative contributions of each of these factors in membrane bending remain unclear. Using the Sar1 protein monomer bound to different nucleotides (GDP vs. GTP) and its GTP-bound dimeric form as examples, we explicitly show that while the precise amino acid sequence and insertion depth of the amphipathic helix are relevant, the generated membrane curvature is most correlated with the volume of protein insertion into the membrane.