Loo Tip W, Clarke David M
Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Biochem Biophys Res Commun. 2016 Apr 1;472(2):379-83. doi: 10.1016/j.bbrc.2016.02.124. Epub 2016 Mar 2.
P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.
P-糖蛋白（P-gp）是一种ABC（ATP结合盒）药物泵。ABC蛋白的一个共同特征是它们被组织成两个结构域。每个结构域包含一个跨膜结构域（TMD）和一个核苷酸结合结构域（NBD）。药物底物和ATP分别结合在TMD和NBD之间的界面处。药物转运涉及ATP依赖的向内（开放，NBD相距较远）和向外（关闭，NBD靠在一起）构象之间的构象变化。在去污剂存在下结晶的P-gp呈现开放结构。人P-gp在去污剂中无活性，但加入脂质后可恢复基础ATP酶活性。脂质可能导致结构域关闭，使NBD靠在一起以允许ATP水解。然而，我们发现，在没有脂质的情况下将结构域交联在一起并不会激活ATP酶活性，这表明脂质可能诱导ATP酶活性所需的其他结构变化。然后，我们测试了脂质对细胞内环4（TMD2）和NBD1之间第一个传输界面处突变体二硫键交联的影响。突变体L443C/S909C和L443C/R905C，但不是G471C/S909C和V472C/S909C，在膜中时可与氧化剂交联。然后将突变体纯化，并在有或没有脂质的情况下进行交联。突变体G471C/S909C和V472C/S909C仅在没有脂质的情况下交联，而突变体L443C/S909C和L443C/R905C仅在有脂质的情况下交联。结果表明，脂质激活了第一个传输界面处的开关，并且P-gp在去污剂和脂质中的结构不同。
