Lipid translocation from one lipid bilayer leaflet to the other, termed flip-flop, is required for the distribution of newly synthesized phospholipids during membrane biogenesis. However, a dedicated biogenic lipid flippase has not yet been identified. Here, we show that the efficiency by which model transmembrane peptides facilitate flip of reporter lipids with different headgroups critically depends on their content of helix-destabilizing residues, the charge state of polar flanking residues, and the composition of the host membrane. In particular, increased backbone dynamics of the transmembrane helix relates to its increased ability to flip lipids with phosphatidylcholine and phosphatidylserine headgroups, whereas a more rigid helix favors phosphatidylethanolamine flip. Further, the transmembrane domains of many SNARE protein subtypes share essential features with the dynamic model peptides. Indeed, recombinant SNAREs possess significant lipid flippase activity.
a b s t r a c tIt has been suggested that lipids translocate between the outer and inner leaflets of fusing membranes, or flip-flop, to facilitate changes in bilayer leaflet areas at various stages of fusion. Here, we investigated the lipid flip activity of synthetic peptides that mimic SNARE transmembrane domains (TMDs). These peptides indeed induce flip of marker lipids. However, mutations that reduce flip activity do not diminish fusogenicity and cholesterol blocks flip much more efficiently than fusion. Therefore, our data do not support a role for flip in membrane fusion. On the other hand, the ability of SNARE TMDs to catalyze flip is consistent with a role of SNAREs in biogenic lipid flip.
Integral membrane transport proteins are essential to life of all organisms from bacteria to mammals. These proteins control the transport of substrates and secondary active transport (SAT) proteins transport a large substrate with the help of the smaller primary substrate (ions or proton). Although crystal structures of these SAT proteins are available, nearly all of these proteins are crystallized in a single state of the transport cycle. Our work aims to use atomic-level simulations to probe these unknown states based on a single crystal structure. The method uses a two-step hybrid simulation approach that involves self-guided Langevin dynamics (SGLD) simulations to enhance conformational sampling in an implicit bilayer and then molecular dynamics (MD) in an explicit bilayer. This method has been shown to be successful for lactose permease (LacY) of E. coli and agrees with many experimental observables, e.g., DEER, FRET, accessibility, etc. (Pendse et al., JMB, 2010). The sodium-hydantoin transporter (Mhp1) is one of the few SAT proteins with conformations in several states of the transport cycle. Mhp1 offers a direct way to compare our new method to enhance SAT protein conformational changes and probe substrate transport. Our method has been successful in switching from the inward-facing and the occluded state to the extracellular (EC) open state. The occluded simulation switched from an EC-closed to EC-open (5.0 to 11 Å) and the intracellular (IC) gate sampled a closed position of 6.050.6Å. A similar outward-facing structure was obtained for the simulation that started in the inward-facing state. The rigid helix bundle, hash motif, and thin gates of Mhp1 all overlay the outward-facing crystal structure. These results show more motions than any previous simulation (occluded or inward-facing) and demonstrate that our method agrees with the 'native' outward-facing x-ray crystal structure.
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