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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.

SUBMITTER: Gustot A

PROVIDER: S-EPMC2863179 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Lipid composition regulates the orientation of transmembrane helices in HorA, an ABC multidrug transporter.

Gustot Adelin A Smriti Ruysschaert Jean-Marie JM McHaourab Hassane H Govaerts Cédric C

The Journal of biological chemistry 20100311 19

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 hydr ...[more]

PMID: 20223819

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Project description:To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine approximately 6:4 POPC:cholesterol<POPC approximately dioleoylphosphatidylcholine (DOPC)<1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG)</=1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.

Project description:BackgroundSince membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle.ResultsHere, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32˚. More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05˚, which is better than 71.47˚ obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew's correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at http://bio-cluster.iis.sinica.edu.tw/TMexpo.ConclusionsTMexpo can better predict rASA and rotational angles than the compared methods. When rotational angles can be accurately predicted, free modeling of transmembrane protein structures in turn may benefit from a reduced complexity in ensembles with a significantly less number of packing arrangements. Furthermore, sequence-based prediction of both rotational angle and lipid exposure can provide essential information when high-resolution structures are unavailable and contribute to experimental design to elucidate transmembrane protein functions.

Dataset Information

Lipid composition regulates the orientation of transmembrane helices in HorA, an ABC multidrug transporter.

Publications

Lipid composition regulates the orientation of transmembrane helices in HorA, an ABC multidrug transporter.

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