Open AccessThe nucleotide‐binding domains of P‐glycoprotein
Author(s) -
Berridge Georgina,
Walker Jennifer A.,
Callaghan Richard,
Kerr Ian D.
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03514.x
Subject(s) - atp binding cassette transporter , nucleotide , biochemistry , cysteine , transporter , cyclic nucleotide binding domain , plasma protein binding , biology , walker motifs , atp hydrolysis , chemistry , enzyme , gene , atpase
The two nucleotide‐binding domains (NBDs) of a number of ATP‐binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co‐operativity has been determined for the NBDs of the human multidrug transporter, P‐glycoprotein. However, the issue of functional symmetry in P‐glycoprotein remains contentious. To address this, the NBDs of P‐glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose‐binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker‐A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time‐dependent, covalent labelling with N ‐ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N ‐ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.