Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues.
It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Gregory
G
Tombline
Lori A
LA
Bartholomew
Grace A
GA
Tyndall
Khursheed
K
Gimi
Ina L
IL
Urbatsch
Alan E
AE
Senior
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics*; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001667:Binding, Competitive; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D050356:Lipid Metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D009154:Mutation; D010843:Pichia / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002264:Carboxylic Acids; D001224:Aspartic Acid; D018698:Glutamic Acid; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008239:Lysine; D000409:Alanine; D004229:Dithiothreitol