de Foresta Béatrice, Vincent Michel, Gallay Jacques, Garrigos Manuel
CEA, iBiTecS, SB(2)SM, CNRS, URA 2096, F-91191, Gif-sur-Yvette, France.
Biochim Biophys Acta. 2010 Mar;1798(3):401-14. doi: 10.1016/j.bbamem.2009.11.019. Epub 2009 Dec 11.
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.
人类多药耐药相关蛋白1（hMRP1/ABCC1）属于ATP结合盒转运蛋白超家族。hMRP1与P-糖蛋白（ABCB1）和乳腺癌耐药蛋白（BCRP/ABCG2）一起，赋予对大量结构多样药物的耐药性。目前hMRP1的拓扑模型包括两个胞质核苷酸结合结构域和17个假定的跨膜（TM）螺旋，形成三个跨膜结构域。诱变和标记研究表明TM16和TM17对功能很重要。我们将TM16片段插入十二烷基磷酸胆碱（DPC）或正十二烷基-β-D-麦芽糖苷（DM）胶束中作为膜模拟物，并扩展了我们之前关于TM17的研究（Vincent等人，2007年，《生物化学与生物物理学报》1768卷，538页）。我们合成了TM16和TM17，其中TM16中的Trp残基W1198和TM17中的W1246作为内在荧光探针，以及TM16和TM17的Trp变体，以探测肽序列中的不同位置。我们通过评估此类相互作用导致的荧光强度增加来评估肽与膜模拟物的相互作用。在所有与胶束结合的肽中，色氨酸残基平均似乎位于胶束头部区域，如其荧光光谱所示。如荧光猝灭所示，每个色氨酸残基对丙烯酰胺和两种DM类似物的溴化酰基链都部分可及。发现色氨酸荧光寿命取决于色氨酸残基在各种肽中的位置，这可能反映了局部结构的差异。远紫外圆二色光谱表明TM16含有显著的β-链结构。连同高Trp相关时间，这些结构的存在表明TM16可能在界面处发生自缔合。总之，这项实验研究表明TM16和TM17在膜模拟物中位于界面处。就hMRP1的整体结构而言，这些TM实验证明的两亲性特性与目前公认的结构模型所暗示的在至少部分亲水转运孔内衬中的作用一致最终结构会因与其他TM螺旋的相互作用而改变。
