Sharma S, Rose D R
Division of Molecular and Structural Biology, Ontario Cancer Institute, University of Toronto, Canada.
J Biol Chem. 1995 Jun 9;270(23):14085-93. doi: 10.1074/jbc.270.23.14085.
Multidrug-resistant tumor cells overexpress P-glycoprotein (170 kDa), a member of the ABC (ATP Binding Cassette)-transporter superfamily. P-glycoprotein has been implicated in transport of a broad range of amphiphilic, hydrophobic drugs from tumor cells. The sequence and structural organization of P-glycoprotein, which consists of 12 transmembrane helices and two cytoplasmic nucleotide binding domains, is similar to other ABC-transporters. It is believed that the nucleotide binding domains of various ABC transporters, which have 30-50% sequence identity, play an important role in coupling ATP hydrolysis to the transport process. To allow structure-function studies of the nucleotide binding domains, the carboxyl-terminal nucleotide binding domain (NBD) of Chinese hamster P-glycoprotein has been cloned, overexpressed, and purified both by itself and as a fusion with maltose-binding protein. It has been demonstrated that the carboxyl-terminal NBD, when overexpressed in Escherichia coli in the absence of transmembrane helices, has very low ATPase activity. This suggests that the amino-terminal nucleotide binding domain and possibly interaction with the transmembrane domains may be required for full ATPase activity. It is also consistent with the idea that the ATPase activity of P-glycoprotein is stimulated in the presence of drugs. Circular dichroism spectral analysis and the ability of carboxyl-terminal NBD, both by itself and as a fusion with maltose-binding protein, to bind ATP-agarose beads and P-glycoprotein specific monoclonal antibodies suggests that the polypeptide folds into a functional domain. Gel filtration chromatography and cross-linking studies indicate that the carboxyl-terminal NBD has a tendency to self-associate to form oligomers. It is speculated that the carboxyl-terminal NBD may play a role in self-association of P-glycoprotein molecules in the plasma membrane.
多药耐药肿瘤细胞过度表达P-糖蛋白（170 kDa），它是ABC（ATP结合盒）转运蛋白超家族的成员。P-糖蛋白与多种两亲性、疏水性药物从肿瘤细胞的转运有关。P-糖蛋白由12个跨膜螺旋和两个胞质核苷酸结合结构域组成，其序列和结构组织与其他ABC转运蛋白相似。据信，各种ABC转运蛋白的核苷酸结合结构域具有30%-50%的序列同一性，在将ATP水解与转运过程偶联中起重要作用。为了对核苷酸结合结构域进行结构-功能研究，中国仓鼠P-糖蛋白的羧基末端核苷酸结合结构域（NBD）已被克隆、过表达，并单独以及与麦芽糖结合蛋白融合表达后进行了纯化。已经证明，当在没有跨膜螺旋的情况下在大肠杆菌中过表达时，羧基末端NBD的ATP酶活性非常低。这表明完整的ATP酶活性可能需要氨基末端核苷酸结合结构域以及可能与跨膜结构域的相互作用。这也与药物存在时P-糖蛋白的ATP酶活性受到刺激的观点一致。圆二色光谱分析以及羧基末端NBD单独以及与麦芽糖结合蛋白融合表达后与ATP-琼脂糖珠和P-糖蛋白特异性单克隆抗体结合的能力表明该多肽折叠成一个功能结构域。凝胶过滤色谱和交联研究表明羧基末端NBD有自我缔合形成寡聚体的倾向。据推测，羧基末端NBD可能在质膜中P-糖蛋白分子的自我缔合中起作用。
