Vincent Michel, Gallay Jacques, Jamin Nadège, Garrigos Manuel, de Foresta Béatrice
CNRS UMR8619 IBBMC, Orsay, F-91405, France; Univ Paris-Sud, Orsay, F-91405, France.
Biochim Biophys Acta. 2007 Mar;1768(3):538-52. doi: 10.1016/j.bbamem.2006.11.021. Epub 2006 Dec 16.
The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala(1227) to Ser(1251)), which contains a single Trp residue (W(1246)) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala(1227) was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring ( approximately 50% alpha-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp(1246) to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including lambda(max), lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.
人类多药耐药蛋白MRP1（或ABCC1）是ABC转运蛋白大家族中最重要的成员之一，在生物学（组织防御）和药理学功能方面均如此。许多研究对MRP1的功能进行了探究，但关于该蛋白的结构数据仍然匮乏。我们研究了预测的跨膜片段17（TM17，从Ala(1227)至Ser(1251)）的结构和动力学，该片段包含一个参与MRP1底物特异性和转运功能的单个Trp残基（W(1246)）。我们合成了TM17以及一个将Ala(1227)替换为带电荷的Lys残基的修饰肽。这两种肽都能很容易地溶解在作为膜模拟物的十二烷基麦芽糖苷（DM）或十二烷基磷酸胆碱（DPC）胶束中。通过稳态和时间分辨Trp荧光光谱研究了这些肽与DM或DPC胶束的相互作用，包括用丙烯酰胺或DM的两种溴化类似物淬灭Trp的实验。通过圆二色性确定了这些肽的二级结构。总体而言，与缓冲液相比，在存在DM或DPC胶束的情况下，获得的结果表明TM17具有显著的结构（约50%的α-螺旋）。基于Trp(1246)对DM的溴化烷基链和/或丙烯酰胺具有显著的可及性，提出了TM17的主要界面位置。将包括λ(max)、寿命分布和Trp旋转流动性在内的各种荧光参数与在相同去污剂中为模型荧光跨膜螺旋测定的参数进行比较，也与TM17的界面位置一致。因此，我们认为TM17的固有特性可能不足以使其如MRP1共有拓扑模型所提出的那样进行跨膜插入。这种插入也可能受其他限制因素控制，如与其他跨膜结构域的相互作用及其在蛋白质序列中的位置。这种预测的跨膜肽的特定行为模式可能是参与底物转运的片段的标志。
