Abstract
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.
Highlights
We have proposed that ATP initially binds to each of the nucleotide binding domains (NBDs) while Pgp is in the open conformation, and the NBDs approach each other to form an integrated structure, the closed conformation, in which catalytic side chains required to stabilize the ATP hydrolysis transition state complex are made available from both NBDs
Both E552Q/E1197Q and E552A/E1197A mutants were able to bind MgATP and MgADP with high affinity, suggesting that they were trapped in the closed conformation of Pgp, which, we propose, occurs immediately before the hydrolytic step
Contrary to previous results [28, 31] we found that both E552Q and E1197Q mutants undergo multiple turnovers during the time course of the assay
Summary
P-glycoprotein; NBD, nucleotide binding domain; Vi, orthovanadate; DTT, dithiothreitol; ABC, ATPbinding cassette. Immediately adjacent to and downstream of the conserved “Walker B” Asp residue might provide the same catalytic carboxylate function in ABC transporter NBDs. A mutagenesis screen of carboxylate residues by Urbatsch et al [28] underscored the importance of the highly conserved, putative catalytic carboxylate residues Glu-552 and Glu-1197 in mouse MDR3 Pgp. Mutagenesis of either residue to Gln yielded Pgp with strongly impaired drug transport and ATPase activities, mutant proteins did show orthovanadate (Vi)-induced trapping of 8-azido-ADP upon incubation with 8-azido-ATP and Vi, suggesting that at least one turnover of 8-azido-ATP hydrolysis as far as the Pi release step was occurring. Using a sensitive radioactive assay, we found that the mutants displayed real but very low ATPase activity and that inability to form the normal transition state, rather than slow ADP release, was the primary defect Both E552Q/E1197Q and E552A/E1197A mutants were able to bind MgATP and MgADP with high affinity, suggesting that they were trapped in the closed conformation of Pgp, which, we propose, occurs immediately before the hydrolytic step (see above). We combined the mutations S528A and S1173A, involving mutations of the conserved Ser residue in the “LSGGQ” ABC signature sequence of NBD1 or NBD2, with the catalytic carboxylate E552A mutation of NBD1 to assess the effects of mutations “in cis” or “in trans” at the putative NBD dimer interface
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