van Wonderen Jessica H, McMahon Róisin M, O'Mara Megan L, McDevitt Christopher A, Thomson Andrew J, Kerr Ian D, MacMillan Fraser, Callaghan Richard
Henry Wellcome Unit for Biological EPR, School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK.
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.
FEBS J. 2014 May;281(9):2190-2201. doi: 10.1111/febs.12773. Epub 2014 Apr 1.
Understanding the process that underlies multidrug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data have recently become available for the murine and Caenorhabditis elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions, these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of EPR spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2 ) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide-bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalization of the data with molecular dynamic simulations indicates that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation.
了解P-糖蛋白（ABCB1）介导的多药识别和外排过程仍然是一项关键的生物学挑战。最近已获得ABCB1的小鼠和秀丽隐杆线虫同源物的结构数据；然而，所有结构都是在没有核苷酸的情况下获得的。这些结构的一个特点是存在一个从蛋白质细胞外表面无法进入的中央腔。为了确定该区域的构象动力学，将跨膜螺旋TM6（331、343和354）和TM12（980）中的几个残基突变为半胱氨酸。根据结构预测，这些残基被认为位于中央腔的内衬或附近。用顺磁探针标记突变体同工型，从而能够应用电子顺磁共振光谱方法。在存在疏水（O2）或亲水（NiEDDA）猝灭剂的情况下记录功率饱和电子顺磁共振光谱，以研究每个残基的局部环境。ABCB1被捕获在其核苷酸结合构象和水解后构象中，并在存在和不存在猝灭剂的情况下再次记录电子顺磁共振光谱。电子顺磁共振谱线形状提供了有关ATP水解过程中这些残基在TM6和TM12内移动的信息。用分子动力学模拟对数据进行合理化分析表明，核苷酸结合后，中央腔转变为向水相开放的构型，从而表明在转运过程中膜两侧交替进入中央腔。
