ABCMdb

ABCB1 p.Glu556Ala

None has been submitted yet.

Publications

PMID: 12437356 [PubMed] Sauna ZE et al: "Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1)."

No. Sentence Comment

2 The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Login to comment
4 Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Login to comment
32 In this study, in addition to the mutants E556Q and E1201Q we have also characterized the E556A and E1201A as well as the double (E556/1201Q and E556/1201A) mutant Pgps. Login to comment
54 The coding sequence for the E556Q mutant primer was 5'-ATC CTC CTG CTG GAT CAG GCC ACG TCA GCC TTG-3'; for the E1201Q mutant primer, 5'-ATT TTG CTT TTG GAT CAA GCC ACG TCA GCT CTG-3'; for the E556A mutant primer, 5'-ATC CTC CTG CTG GAT GCG GCC ACG TCA GCC TTG-3'; and for the E1201A primer, 5'-ATT TTG CTT TTG GAT GCA GCC ACG TCA GCT CTG-3'. Login to comment
65 Crude membranes were prepared from vTF7-3 infected HeLa cells transfected with vector pTM1-MDR1 wild type, pTM1-MDR1-E556Q, pTM1-MDR1-E1201Q, pTM1-MDR1-E556/1201Q, pTM1-MDR1-E556A, pTM1-MDR1-E1201A, and pTM1-MDR1- E556/1201A as described previously (28, 31) and stored at -70 °C. Total protein was quantified by the Amido Black protein estimation method as previously described (32), and Pgp expression level was determined by immunoblot analysis using the monoclonal antibody C219 (30, 33). Login to comment
279 To assess whether a Glu to Ala substitution will affect the cleavage of the -γ-phosphate bond of ATP, we generated the mutant Pgps where Glu was replaced with Ala (E556A, E1201A, and E556/1201A). Login to comment
280 Characterization of these mutants showed that (1) they exhibited comparable cell surface expression, (2) similar to E f Q (see Figure 1A), E f A substitutions also resulted in loss of transport activity, and (3) the single mutants E556A and E1201A, similar to E556Q and E1201Q (see Figure 1C), showed some trapping of [R-32 P]-8-azidoADP in the absence of Vi, which was enhanced in the presence of 0.25 mM Vi (data not shown). Login to comment
294 Human Pgp mutants of the conserved glutamate residue in the Walker B region (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) showed cell surface expression levels comparable to that of the wild-type protein, but the transport function was abrogated in all of the mutant Pgps (Figure 1A,B; data for E556A, E1201A, and E556/1201A not shown). Login to comment
333 The single mutants (E556Q, E556A, E1201Q, and E1201A), on the other hand, show normal release of ADP and can bind ATP during next step but exhibit greatly reduced ability to hydrolyze it (see Figure 2A, panels I-III; data with E556A and E1201A not shown). Login to comment

PMID: 14576852 [PubMed] Ambudkar SV et al: "P-glycoprotein: from genomics to mechanism."

No. Sentence Comment

276 Adapted from Sauna et al. (2001a, b) Furthermore, the equivalent mutations in human P-gp, E556Q, E556A, E1201Q, E1201A, and the double mutants E556Q/E1201Q and E556A/E1201A, all allow for normal levels of Vi-dependent [a-32 P]8-azidoADP trapping, and the trapped nucleotide has been demonstrated to be [a-32 P]8-azidoADP (Sauna et al., 2002). Login to comment
277 Thus, the substitutions of the residues E556 and E1201 with Q or A in human P-gp do not block hydrolysis of ATP per se. Interestingly, the double mutants E556Q/ E1201Q and E556A/E1201A show trapping of [a-32 P]8-azidoADP even in the absence of Vi. Login to comment
279 These double mutants of P-gps (E556Q/E1201Q and E556A/E1201A) thus provide an independent validation that the Vi-trapped transition state of P-gp is indeed a 'true` transition state, and they provide an interesting system where one can obtain the transition state of P-gp in the absence of agents such as Vi or beryllium fluoride. Login to comment
326 The single mutants (E556Q, E556A, E1201Q, and E1201A) on the other hand show normal release of ADP, and can bind ATP during next step, but are unable to hydrolyse it. Login to comment

PMID: 14596601 [PubMed] Carrier I et al: "Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein."

No. Sentence Comment

229 Moreover, in their recent work, Sauna and colleagues also demonstrated using Rand γ-labeled 8-azido-[R-32 P]ATP that mutants at the equivalent positions of the human MDR1 protein (E556Q and E556A, E1201Q and E1201A, and the double mutants) are indeed capable of ATP hydrolysis and single-site catalysis (59). Login to comment
234 Indeed, they suggest that the single E556A/Q and E1202A/Q mutants are defective in the second ATP hydrolysis event of the catalytic cycle, which should reset the protein after hydrolysis at the first site. Login to comment

PMID: 15159388 [PubMed] Tombline G et al: "Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly."

No. Sentence Comment

48 Two such mutants have been reported, namely the E556Q/E1201Q and E556A/E1201A mutants of human Pgp (28). Login to comment

PMID: 16352426 [PubMed] Ambudkar SV et al: "The power of the pump: mechanisms of action of P-glycoprotein (ABCB1)."

No. Sentence Comment

53 Hoof et al. (1994) Human L531R Decreased cell surface expression Bakos et al. (1997) G534V K536I Normal cell surface expression K536R Normal ATP hydrolysis I541T R543S LSGGQ or linker peptide or signature motif Human R538M Normal cell surface expression Decreased ATP hydrolysis Bakos et al. (1997) I541R Normal cell surface expression No ATP hydrolysis Walker B Mouse D551N D1196N No ATP hydrolysis, required for Mg2+ binding Urbatsch et al. (1998) Human D555A D1200A Same as above Hrycyna et al. (1999) Walker B Mouse E552A E1197A Trapping of ATP, no steady-state hydrolysis Tombline et al. (2004b) Mouse E552Q E1197Q No steady-state ATP hydrolysis Vigano et al. (2002) Human E556A E1201A Trapping of ATP or ADP in the absence of vanadate, low levels of ATP hydrolysis Sauna et al. (2002) D-loop Mouse D558N D1203N Decreased ATP hydrolysis Urbatsch et al. (2000b) the ABC transporter superfamily. Login to comment