Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants |
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Authors: | Bé atrice de Foresta,Michel Vincent,Manuel Garrigos |
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Affiliation: | a CEA, iBiTecS, SB2SM, F-91191, Gif-sur-Yvette, France b CNRS, URA 2096, F-91191, Gif-sur-Yvette, France c Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université Paris-Sud, UMR8619-CNRS, IFR115, F-91405 Orsay, France |
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Abstract: | The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices. |
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Keywords: | hMRP1 (or ABCC1), human multidrug resistance protein 1 BCRP (or ABCG2), breast cancer resistance protein LTC4, cysteinyl leukotriene C4 E217βG, estradiol 17-(β- smallcaps" >d-glucuronide) GSH, reduced glutathione DM, n-dodecyl-β- smallcaps" >d-maltoside BrDM, 7, 8-dibromododecylmaltoside BrUM, 10, 11-dibromoundecanoylmaltoside DPC, dodecylphosphocholine cmc, critical micellar concentration NATA, N-acetyltryptophanamide TOE, tryptophan octyl ester DMSO, dimethylsulfoxide TFE, trifluoroethanol TFA, trifluoroacetic acid MSD, membrane-spanning domain TM, transmembrane fragment NBD, nucleotide-binding domain MEM, maximum entropy method CD, circular dichroism FWHM, full-width at half-maximum P3, K2WL9AL9K2A P5, K2CLWL7AL9K2A P7, K2CL3WL5AL9K2A P9, K2CL5WL3AL9K2A P11, K2CL7WLAL9K2A P13, K2CL9WL9K2A TM16, A1195NRWLAVRLECVGNCIVLFAALFAV1219 mTM16, A1195NRWLAVRLESVGNSIVLFAALFAV1219 W19-mTM16, A1195NRYLAVRLESVGNSIVLWAALFAV1219 TM17, A1227GLVGLSVSYSLQVTTYLNWLVRMS1251 mTM17, K1227GLVGLSVSYSLQVTTYLNWLVRMS1251 W10-mTM17, K1227GLVGLSVSWSLQVTTYLNYLVRMS1251 |
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