Both ATP sites of human P-glycoprotein are essential but not symmetric.
Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.
Hrycyna
C A
CA
Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892, USA.
Ramachandra
M
M
Germann
U A
UA
Cheng
P W
PW
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000187:drug effects / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001386:Azides / Q000037:antagonists & inhibitors / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D001831:Body Temperature; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D016384:Consensus Sequence / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005615:Freezing; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D017354:Point Mutation; D011224:Prazosin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D001386:Azides; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; C041552:azidoprazosin; D014700:Verapamil; D000251:Adenosine Triphosphatases; D011224:Prazosin