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首页> 外文期刊>The FEBS journal >The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis
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The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis

机译：ABCB1的中央腔在ATP水解过程中经历交替进入

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摘要

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 Caenorhabditiselegans 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 (O-2) 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
机译：了解P-糖蛋白（ABCB1）构成多药识别和外排基础的过程仍然是关键的生物学挑战。最近，已有关于ABCB1的鼠和秀丽隐杆线虫同系物的结构数据。然而，所有结构都是在没有核苷酸的情况下获得的。这些结构的一个特征是存在中央腔，该中央腔从蛋白质的细胞外表面无法进入。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，建议将这些残基排列在中心腔中或位于中心腔附近。突变体同工型用顺磁性探针标记，可以应用EPR光谱方法。在疏水性（O-2）或亲水性（NiEDDA）淬灭剂存在下记录功率饱和EPR光谱，以研究每个残基的局部环境。将ABCB1捕获在其核苷酸结合构象和水解后构象中，并在存在和不存在淬灭剂的情况下再次记录EPR光谱。 EPR线形可提供有关ATP水解过程中TM6和TM12中这些残基运动的信息。用分子动力学模拟对数据进行合理化表明，核苷酸结合后，空腔被转换为对水相开放的构型，从而表明在移位过程中交替进入膜相反侧的空腔

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