Both ATP Sites of Human P-Glycoprotein Are Essential but Not Symmetric

Abstract

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

Conserved Walker A Ser Residues in the Catalytic Sites of P-glycoprotein Are Critical for Catalysis and Involved Primarily at the Transition State Step

“…Therefore, in this work, the two nucleotide sites behaved symmetrically. Other workers have suggested that the two sites might behave asymmetrically (43).…”

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

“…When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities. Functional coupling of ATPase activities observed for P-glycoprotein and MalK might arise if ATP hydrolysis is required at two distinct steps during a single turnover of the catalytic cycle as has been recently suggested for P-glycoprotein (40,41). Activation of the ATPase activity at each NBD might require transient conformational states that are acquired at specific steps of a translocation cycle.…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…For some transporters, the decision may be stochastic (i.e. for P-gp; [63,65]). However, for both MRP1 [66] and SUR1 (ABCC8; [67]), there is evidence that NBD1 provides the initial ATPbinding site.…”

“…Replacement of a single amino acid R697A at the NBD dimer interface, which interacts with the opposing NBD, abolishes alternating hydrolysis and reduces the efficiency of DNA repair [36]. For some ABC transporters, the 'choice' of which NBD hydrolyses first may be stochastic [65]. For others, there is a clear asymmetry: for MRP1, NBD2 appears to hydrolyse ATP initially [66,74], and in CFTR, hydrolysis of one ATP is much slower than the other [84].…”