Delannoy Sabine, Urbatsch Ina L, Tombline Gregory, Senior Alan E, Vogel Pia D
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
Biochemistry. 2005 Oct 25;44(42):14010-9. doi: 10.1021/bi0512445.
Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.
利用自旋标记的三磷酸腺苷（ATP）进行电子自旋共振（ESR）光谱分析，以研究核苷酸与多药耐药性P-糖蛋白（P-gp）的结合以及其结构转变。观察到自旋标记连接在核糖上的自旋标记ATP（SL-ATP）是P-gp的一种优良底物类似物。SL-ATP在药物刺激下以约正常ATP水解速率14%的速度被水解，并使酶在过渡态和基态下实现可逆捕获。在存在Mg2+但不存在维拉帕米等转运底物的情况下，观察到每个P-gp总共结合两个核苷酸，结合亲和力为366微摩尔。在存在维拉帕米的情况下，SL-ATP与野生型P-gp的结合导致蛋白质结合的自旋标记部分减少，这很可能是由于P-gp内的构象转变，使半胱氨酸靠近自旋标记，从而使自由基发生化学还原。我们通过使用一种P-gp突变体解决了这个问题，该突变体中所有天然存在的半胱氨酸都被丙氨酸取代。SL-ATP与这种突变型P-gp的平衡结合导致两个核苷酸的最大结合；在不存在维拉帕米时结合亲和力为223微摩尔，在存在维拉帕米时为180微摩尔。在存在SL-ATP的情况下，野生型和无半胱氨酸P-gp的相应ESR光谱表明，P-gp的一个半胱氨酸侧链位于结合核苷酸的核糖附近。将SL-ATP捕获为AlF(x)加合物会产生ESR光谱，显示自由基强烈固定，支持P-gp在其过渡态形成封闭构象。这项研究首次采用ESR光谱结合自旋标记核苷酸类似物来研究P-糖蛋白。该研究表明，SL-ATP是一种优良的底物类似物，将有助于进一步探索这种重要酶的核苷酸结合域内的结构和动力学。
