Effect of anionic lipids on ABC transporters

Abstract

The role of ATP-Binding Cassette (ABC) transporters in multidrug and agrochemical resistance has been a rising concern across medicine and agriculture. One way to tackle such resistance would be to target the interactions of lipids in the bilayer with the transmembrane helices of the transporters. As a prelude to such studies, it is first necessary to identify specific lipid binding sites at the membrane lipid interface that modify transport activity and could serve as potential inhibitor targets. In this study, the difference in the effects of anionic, neutral and cationic phospholipids has been demonstrated through the varying ATPase activity of Sav1866 and McjD. Furthermore, specific binding sites for anionic phospholipids that contain a cluster of positively charged residues on the cytoplasmic side of McjD was revealed using fluorescence quenching technique. Upon removal of the positive residues, the binding affinity for the anionic lipids was lost. Due to the nature of the technique, only a single tryptophan residue on the protein was required. Therefore, a systematic study comprising of site-directed mutagenesis was used to find the optimal replacement for the endogenous tryptophans to produce an active mutant. Subsequent inactive mutants were characterised using a crosslinking technique to compare their conformational status with the wild-type protein. Another aspect of this study was to purify Sav1866 and McjD using detergent-free styrene maleic acid (SMA) to preserve their native environment, which is often disturbed by using detergents. The SMA purification generated pure protein with a comparable yield to the detergent purification. Lastly, as part of the CASE studentship with Syngenta, a eukaryotic ABC transporter, Mdl1 was purified and characterised. The work produced was at its preliminary stage en route to elucidating its mechanism in fungicide resistance.

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