TAILIEUCHUNG - Báo cáo khoa học: Functional symmetry in the isolated domain demonstrated by N-ethylmaleimide labelling

The twonucleotide-bindingdomains (NBDs)of anumberof 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 trans-porter, P-glycoprotein. However, the issue of functional symmetry in P-glycoprotein remains contentious. | Eur. J. Biochem. 270 1483-1492 2003 FEBS 2003 doi The nucleotide-binding domains of P-glycoprotein Functional symmetry in the isolated domain demonstrated by Methylmaleimide labelling Georgina Berridge1 Jennifer A. Walker1 Richard Callaghan1 and Ian D. Kerr1 2 1Nuffield Department of Clinical Laboratory Sciences University of Oxford John Radcliffe Hospital Oxford UK 2School of Biomedical Sciences University of Nottingham Queen s Medical Centre Nottingham UK 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 timedependent 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. Keywords ABC transporter cysteine functional symmetry maleimide Walker-A. ATP-binding cassette ABC transporters are multidomain membrane proteins responsible for the controlled efflux and

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