title: 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.
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-beta-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 beta-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.
authors.affiliation: CEA, iBiTecS, SB(2)SM, CNRS, URA 2096, F-91191, Gif-sur-Yvette, France. Beatrice.de-foresta@cea.fr
authors.forename: Béatrice
authors.initials: B
authors.lastname: de Foresta
authors.forename: Michel
authors.initials: M
authors.lastname: Vincent
authors.forename: Jacques
authors.initials: J
authors.lastname: Gallay
authors.forename: Manuel
authors.initials: M
authors.lastname: Garrigos
mesh_terms: D020106:Acrylamide / Q000494:pharmacology; D000595:Amino Acid Sequence; D016880:Anisotropy; D002021:Buffers; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002942:Circular Dichroism; D003470:Culture Media; D005960:Glucosides / Q000378:metabolism; D054879:Halogenation / Q000187:drug effects; D006801:Humans; D008823:Micelles; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D017186:Titrimetry; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D020106:Acrylamide; D002021:Buffers; D003470:Culture Media; D005960:Glucosides; D008823:Micelles; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D010767:Phosphorylcholine; D014364:Tryptophan; C028810:dodecylphosphocholine; C040358:dodecyl maltoside; C421109:multidrug resistance-associated protein 1