Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway.
We found recently that the combined mutation of both "catalytic carboxylate" residues (E552A/E1197A) in mouse P-glycoprotein (Pgp) arrested the protein in an "occluded nucleotide conformation", possibly a stabilized dimer of nucleotide-binding domains (NBDs), that binds MgATP tightly at stoichiometry of 1 mol/mol Pgp [Tombline, G., Bartholomew, L., Urbatsch, I. L., and Senior, A. E. (2004) J. Biol. Chem. 279, 31212-31220]. Here, we further examine this conformation in respect to its potential involvement in the catalytic pathway. The occluded nucleotide conformation is promoted by drugs. Verapamil markedly accelerated the rate of tight binding of MgATP, whereas it did not effect the rate of dissociation. Mutations in "Q-loop" residues that are thought to interfere with communication between drug and catalytic sites prevented the occluded nucleotide conformation, as did covalent reagents N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, which are known to inhibit ATP hydrolysis by reacting in catalytic sites. Mutations of Walker A Ser and Lys residues in combination with E552A/E1197A had the same effect, showing that interaction of these conserved residues with MgATP is required to stabilize the occluded nucleotide conformation. We present an enzymatic scheme that incorporates this conformation. We propose that upon initial loose binding of MgATP at two nucleotide-binding domains (NBDs), together with drug binding, the NBDs dimerize to form the occluded conformation, with one tightly bound MgATP committed to hydrolysis. The pathway progresses such that the tightly bound MgATP enters the transition state and is hydrolyzed. This work suggests that small molecules or peptides that interact at the NBD dimer interface might effectively disable Pgp catalysis.
Tombline
Gregory
G
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Box 712 Rochester, New York 14642, USA.
Muharemagić
Alma
A
White
Lori Bartholomew
LB
Senior
Alan E
AE
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D051379:Mice; D009154:Mutation; D019920:Photoaffinity Labels / Q000737:chemistry; D011487:Protein Conformation; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D004791:Enzyme Inhibitors; D019920:Photoaffinity Labels; D018698:Glutamic Acid; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014700:Verapamil; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide