Lipid Composition Regulates the Orientation of Transmembrane Helices in HorA, an ABC Multidrug Transporter

Abstract

ATP-binding cassette (ABC) transporters constitute a large class of molecular pumps whose central role in chemotherapy resistance has highlighted their clinical relevance. We investigated whether the lipid composition of the membrane affects the function and structure of HorA, a bacterial ABC multidrug transporter. When the transporter was reconstituted in a bilayer where phosphatidylethanolamine (PE), the main lipid of the bacterial membrane, was replaced with phosphatidylcholine (PC), ATP hydrolysis and substrate transport became uncoupled. Although ATPase activity was maintained, HorA lost its ability to extrude the prototypical substrate Hoechst33342. Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed that, although the secondary structure of the protein was unaffected, the orientation of the transmembrane helices (TM) was modified by the change in lipid composition. The orientation of the backbone carbonyls indicated that the helices opened wider in PE versus PC-containing liposomes, with 10 degrees difference. This was supported by hydrogen/deuterium exchange studies showing increased protection of the backbone from the solvent in PC-containing liposomes. Electron Paramagnetic Resonance was used to further probe the structural change. In the PC-containing liposomes we observed increased mobility of the spin label in TM4, along with increased exposure to molecular oxygen, used as a hydrophobic quencher. This indicates that the lipid change induced modification of the orientation of TM4, exposing Cys-180 to the lipid phase. The lipid composition of the bilayer thus modulates the structure of HorA, and in turn its ability to extrude its substrates.Over the last thirty years, our vision of the biological membrane has evolved from the fluid mosaic model (1) to that of a remarkably complex system where a myriad of molecules are tightly organized along the membrane plane to properly interact and achieve numerous biological functions.Precise interplay between lipids and proteins must occur to achieve biological function, and membrane proteins have evolved specific sequence motifs to adapt to their environment. Studies have begun to evidence how proteins and lipids interact. It emerges notably that membrane proteins are surrounded by a shell of slow-moving lipids (annular lipids) distinguishable from the bulk phase. Other lipids (non-annular lipids) can achieve tight and specific interactions with a protein and act as cofactors essential to protein function (2, 3). The lipid composition of the membrane can have significant effects on, and even regulate, membrane protein function. In a number of cases, protein function is proposed to be influenced by the bulk physico-chemical properties of the membrane, which in turn are strictly determined by the exact lipid composition of the bilayer (2-4). Such properties include hydrophobic thickness (4), phase transition (5), curvature (6), and lateral pressure (3). It has also been proposed that a specific lipid might act as a molecular cha...

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

“…These are all membrane transport proteins with high degrees of conformational flexibility. In addition, the following transporters from other sources show a requirement for PE after reconstitution into liposomes in order to display uphill transport, which is not satisfied by PC: the multidrug transporter (LmrP) of L. lactis (22,23), the leucine permease of P. aeruginosa (24), the branched chain amino acid transporter of S. cremoris (25), and the ABC transporter HorA from L. lactis (26). Because in all cases the dioleoyl form of PC was used, the lipid dependence for supporting uphill transport needs to be reinvestigated in the above cases using a variety of PC species in vitro and expression in cells containing PC.…”

“…Because phosphatidylserine supports uphill transport and progressive methylation of the amine of PE resulted in a parallel decrease in the level of uphill transport observed in vitro, it was suggested that an ionizable amine was required to support uphill transport (13). PC also did not substitute for PE in proteoliposome studies of proline transport by PutP from E. coli (21), multidrug transport by LmrP from Lactococcus lactis (22,23), leucine transport by the leucine permease from Pseudomonas aeruginosa (24), branched chain amino acid transport by the transporter from Streptococcus cremoris (25), and the ABC transporter HorA from L. lactis (26). The failure of PC to substitute for PE in the case of LacY as well as several other secondary transporters has led to the general conclusion that an ionizable lipid amine may be either involved directly in the transport mechanism or required for proper assembly.…”

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

“…An ionizable amine or hydrogen bond donor capacity is also required for uphill transport because methylation of the amine of PE progressively reduces activity (10,11). PE but not diC 18∶1 PC or eukaryotic-derived PC also supports uphill transport by the multidrug transporter (LmrP) of Lactococcus lactis (12,13), the leucine permease of Pseudomonas aeruginosa (14), the branched chain amino acid transporter of Streptococcus cremoris (15), the ABC transporter HorA from Lactococcus lactis (16), and the Ca 2þ ATPase of the sarcoplasmic reticulum (17).…”

Influence of lipids on protein-mediated transmembrane transport

“…The bacterial ABC multidrug transporter HorA of Lactobacillus brevis required PE for ATPcoupled substrate extrusion from liposomes (Gustot et al, 2010). When PE was replaced by PC, transport activity was abolished even though it hydrolyzed ATP at more than twice the rate than in PE.…”