Lipid-Dependent Alternating Access Mechanism of a Bacterial Multidrug ABC Exporter

Abstract

By undergoing conformational changes, active membrane transporters alternate between an inward-facing (IF) and an outward-facing (OF) state to transport their substrates across cellular membrane. The conformational landscape of membrane transporters, however, could be influenced by their environment, and the dependence of the alternating access mechanism on the lipid composition has not been understood at the molecular level. We have performed an extensive set of microsecond-level all-atom molecular dynamics (MD) simulations on bacterial ATP binding cassette (ABC) exporter Sav1866 in six different phosphocholine (PC) and phosphoethanolamine (PE) lipid membrane environments. This study mainly focuses on the energetically downhill OF-to-IF conformational transition of Sav1866 upon the ATP hydrolysis. We observe that the transporter undergoes large-scale conformational changes in the PE environment, particularly in the POPE lipids, resulting in an IF-occluded conformation, a transition that does not occur when the transporter is embedded in any of the PC lipid bilayers. We propose that the PE lipids facilitate the closing of the protein on the periplasmic side due to their highly polar headgroups that mediate the interaction of the two transmembrane (TM) bundles by a network of lipid-lipid and lipid-protein hydrogen bonds. POPE lipids in particular facilitate the closure of periplasmic gate by promoting a hinge formation in TM helices and an interbundle salt bridge formation. This study explains how the alternating access mechanism and the flippase activity in ABC exporters could be lipid-dependent.

Figure 1

Conformational dynamics of Sav1866 in POPE and POPC lipid environments. (A) Crystal structure of Sav1866 in the OF state: the monomers A and B are colored green and orange, respectively, and the two ATP molecules are colored in magenta. β time series in POPE (B) and POPC (D) simulations along with the β distribution in the last half of the simulations (i.e., from 1.2 to 2.4 μs) for POPE (C) and POPC (E) simulations. (F) Projection of POPE and POPC trajectories onto the (PC1, PC2) space, i.e., the two first principal components of protein C_α atoms. Snapshots of the transporter conformation before and after the equilibration in the POPE (G) and POPC (H) bilayers. Water density in and around the transporter associated with a 10 Å thick cross section of simulation box during the first and last 0.5 μs of POPE1 (I) and POPC1 (J) simulations.

Figure 2

Lipid-mediated periplasmic gate closure. Outer leaflet lipid headgroup occupancy isosurfaces in and around the protein based on POPE1 (A) and POPC1 (B) simulations. (C–E) Stepwise lipid-mediated periplasmic gate closing mechanism of Sav1866 in POPE lipids. TM helices h1 (cyan) and h6 (magenta), which are directly involved in the mechanism, are shown in cartoon representation. Periplasmic residues K38, D42, T276, and T279 are shown in thick licorice representation, while the lipid headgroups and tails are shown in thin ball-and-stick and licorice representations, respectively. Blue dashed lines represent the interbundle, lipid–protein, and lipid–lipid hydrogen bonds. (F) Maximal hydrogen bond occupancy (in percentage) of any single lipid with residues of interest in POPE and POPC simulations. (G) Average number of protein–lipid hydrogen bonds with an occupancy greater than 5% in POPE and POPC simulations. Number of interlipid hydrogen bonds as a function of time formed between the lipids positioned within 8 Å of the periplasmic residues K38, D42, T276, and T279 in POPE1 (H) and any of the POPC (I) simulations. Number of lipid–protein hydrogen bonds as a function of time formed between the lipids and the periplasmic residues K38, D42, T276, and T279 in POPE1 (J) and POPC1 (K) simulations. K38-T279 and D42-T276 hydrogen bond donor–acceptor distance time series for monomer-A in POPE1 (L) and POPC1 (M) simulations. (N, O) Same as parts L and M for monomer-B.

Figure 3

Interactions between protein and lipid tails. Hydrophobic lipid binding pocket in monomer-A (A) and monomer-B (B). (C) TM helices and important residues within the lipid binding pocket of monomer-A. h3, h4, and h6 are colored blue, gray, and magenta, respectively. Key residues of the h6 (L297, F301, L304, and M311) interacting with the lipid tails are represented by orange spheres, and lipids binding in the pocket are shown in the ball-and-stick/licorice representation. Residue R186, which interacts with lipid headgroups, is represented by red spheres. (D) Average contact frequency of POPE and POPC lipids with h6 residues based on all three simulation sets. The contact was defined based on a cutoff distance of 3.0 Å. (E) Average hydrogen bond occupancy (%) of R186 from with the POPE and POPC lipids based on all three simulation sets.

Figure 4

Formation of h3 hinge and R81-D145 salt bridge in POPE lipid membrane. (A) Crystal structure of Sav1866 with the region of interest highlighted by a red circle. (B, C) Two perpendicular views of the highlighted region in part A. Salt bridge forming residues R81, D145, and R296 and their corresponding helices h2, h3, and h6 are highlighted. (D–F) Same as parts A–C using a representative structure of the transporter resulting from POPE simulations. R81-D145 (G) and R296-D145 (H) salt bridge distance time series in POPE simulations. (I) Helical content of the residues 138–141 in monomer-A in POPE simulations. (J–L) Same as parts G–I in POPC simulations.

Figure 5

Sav1866 in non-PO environments. (A) Average β in all PO and non-PO lipid environments based on the last 0.5 μs of the simulations. Projection of DPPE/DPPC (B) as well as DOPE/DOPC (C) trajectories onto their corresponding (PC1,PC2) space. (D) Projection of all PE and PC simulation trajectories (excluding the negative control) onto the (PC1,PC2) space. Representative snapshots of side views of the DOPE (third set, DOPE3; E) and DPPE (first set, DPPE1; F) lipids blocking the protein from closing through interacting with charged residues of the substrate translocation chamber. Positively charged residues (R81, R295, and R296) of the translocation chamber are colored blue, and negatively charged ones (D145 and E288) are colored red. All non-carbon and non-hydrogen atoms of lipids are represented as spheres and carbons as ball and sticks. Oxygen, nitrogen, and phosphorus atoms of lipids are colored red, blue, and gold, respectively. Hydrogen bond interaction occupancies (in percentage) of respective DOPE (G) and DPPE (H) lipids (shown in panels E and F) with the charged residues of the translocation chamber.

Figure 6

Flippase activity of Sav1866. Side views of POPE (A) and DOPE (B) lipids binding parallel to the membrane normal in the substrate translocation chamber. In these two cases the periplasmic gate is closed. (C) Representative snapshot from the third set of DOPE simulations, where the lipid is binding in the translocation chamber. In this case, the periplasmic gate stays open. (D) Tilt angle of the lipids that are shown in parts A–C as a function of time. (E) Distance between the periplasmic residues K38 and T279 as a function of simulation time. (F–H) Hydrogen bond occupancies (in percentage) of the same lipids shown in parts A–C with the charged residues (E288, R295, and R296, respectively) lining the translocation chamber.