title: Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly.
abstract: Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.forename: Lori A
authors.initials: LA
authors.lastname: Bartholomew
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: Alan E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate