Abstract
P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg(2+) in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an "open" catalytic site conformation, where the Ser-OH is dispensable. This changes to a "closed" conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.
Highlights
Pgp is a member of the ABC transporter superfamily (6)
One is the resolved x-ray structure of the nucleotidebinding subunit HisP from the ABC transporter histidine permease (32), which, as we argue in the Introduction, likely provides a reasonable facsimile of the structure of the nucleotide-binding domains of Pgp
We generated mutations S430A, S430T, S1073A, and S1073T in mouse MDR3 Pgp, in order to study the catalytic role of these Ser residues
Summary
P-glycoprotein; DM, n-dodecyl-D-maltoside; Vi, orthovanadate; AlFx, fluoroaluminate; BeFx, beryllium fluoride complex; DTT, dithiothreitol; AMPPNP, adenosine 5Ј-(,␥-imino)triphosphate. It was shown that disruption of just one of the two sites by covalent chemical modification (23) or mutagenesis (24, 25) was sufficient to prevent even a single turnover of ATP hydrolysis at the other, intact site These experiments supported a working model of the catalytic mechanism in which the two sites alternate to hydrolyze ATP (26). The Walker A consensus sequence (GXXXXGK(S/T)), found in numerous ATP- and GTP-hydrolyzing enzymes, ends in a highly conserved Ser or Thr. In cases where x-ray structures are available (e.g. myosin, ATP synthase F1 sector, G-proteins), the hydroxyl oxygen of this residue is seen to provide a direct ligand to the Mg2ϩ of the substrate MgNTP (35–37). To investigate the possible role of Ser-430 and Ser-1073 in Pgp catalysis, and to further study the role of the two catalytic sites, we generated mutations at these positions in mouse MDR3 Pgp. Mutant proteins were expressed in Pichia pastoris and purified to homogeneity (31)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
