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Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both
I L Urbatsch1, L Beaudet, I Carrier
1Department of Biochemistry, McGill University, Montréal, Québec, Canada, H3G 1Y6.
Abstract:
Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.
Insights
Investigating P-glycoprotein (Mdr3) ATP hydrolysis, this study found that mutations in either nucleotide-binding site abolish ATPase activity, demonstrating essential cooperative interactions between both sites for function.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- P-glycoprotein (P-gp), encoded by the multidrug resistance gene (MDR) family, is an ATP-dependent efflux pump crucial for cellular detoxification.
- Mdr3, a specific P-gp isoform in mice, plays a role in phospholipid transport, and its ATPase activity is central to its function.
- The regulation of ATP hydrolysis by P-gp involves two nucleotide-binding (NB) sites, but their precise roles and interactions remain incompletely understood.
Purpose of the Study:
- To elucidate the functional roles of the two NB sites in the ATP hydrolysis regulation of mouse P-glycoprotein (Mdr3).
- To investigate the interdependence and cooperative interactions between the N- and C-terminal NB sites of Mdr3.
- To characterize the impact of specific mutations within the Walker A and B motifs of the NB sites on Mdr3's ATPase activity and function.
Main Methods:
- Overexpression and purification of hexahistidine-tagged mouse Mdr3 in the yeast Pichia pastoris using Ni-affinity chromatography.
- Site-directed mutagenesis to introduce point mutations (K429R, K1072R in Walker A; D551N, D1196N in Walker B) in the conserved motifs of the two NB sites.
- Characterization of ATPase activity in purified Mdr3 mutants using vanadate trapping of 8-azido-nucleotide and photolabeling assays.
Main Results:
- Purified Mdr3 from P. pastoris exhibited high, verapamil-stimulated ATPase activity, confirming its functional integrity.
- Mutations in either the Walker A or B motifs of both NB sites (single and double mutants) resulted in a complete loss of detectable ATPase activity.
- Vanadate-induced trapping and photolabeling with 8-azido-ATP, which requires nucleotide hydrolysis, were abolished in all Mdr3 mutants, indicating severely impaired ATP hydrolysis (<0.3% of wild-type activity).
Conclusions:
- Mutations in one NB site prevent nucleotide turnover and trapping in the other NB site, indicating a lack of independent function.
- The two NB sites of Mdr3 are not functionally distinguishable and require cooperative interactions for ATP hydrolysis.
- These findings highlight the essential interdependence of both nucleotide-binding sites for the catalytic activity of P-glycoprotein.