Cloning, Overexpression, Purification, and Characterization of the Carboxyl-terminal Nucleotide Binding Domain of P-glycoprotein

• Published in: The Journal of biological chemistry Vol. 270; no. 23; pp. 14085 - 14093
• Main Authors: Sharma, Sadhana, Rose, David R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.06.1995; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; ATP Binding Cassette Transporter, Subfamily B, Member 1 - chemistry; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; Base Sequence; Binding Sites; Blotting, Western; Cloning, Molecular; Molecular Sequence Data; Nucleotides - metabolism; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Structure, Secondary; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• 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.