title: Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508.
abstract: Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Gabriel
authors.forename: Janet
authors.initials: J
authors.lastname: Storm
authors.forename: Alice
authors.initials: A
authors.lastname: Rothnie
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Taylor
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Linton
authors.forename: Ian D
authors.initials: ID
authors.lastname: Kerr
authors.forename: Richard
authors.initials: R
authors.lastname: Callaghan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009711:Nucleotides