ABCMdb

ABCB1 p.Tyr401Ala

None has been submitted yet.

Publications

PMID: 16768456 [PubMed] Kim IW et al: "The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette."

No. Sentence Comment

86 HighFive insect cells (Invitrogen) were infected with the recombinant baculovirus carrying the wild type and Y401A, Y1044A, Y401A/Y1044A, Y401C, Y401L, and Y401W mutant human MDR1 cDNAs with a His6 tag at the C-terminal end [BV-MDR1(His6)] as described previously (37). Login to comment
100 The Pgp‚Mg-8-azido[R-32 P]ADP‚ BeFx or Pgp‚Mg-8-azido[R-32 P]ADP‚Vi preor posthydrolysis transition state conformation was generated as described Table 1: Effect of Substitution of the Conserved Y401 and Y1044 Residues in ATP Sites on Pgp Cell Surface Expression, Transport Function, ATP Binding, Nucleotide Trapping, and Hydrolysisa construct cell surface expressionb (%) transport functionc (%) ATP bindingd (%) ADP-Vi trappinge (%) ATP hydrolysisf (%) wild-type MDR1 100 100 100 100 100 Y401W 100 100 90-95 90-95 85-90 Y401F 95-100 90-100 NTg NTg NTg Y401C 90-95 45-55 50 <20 NTg Y401L 95-100 25-30 30-35 20-25 NTg Y401A 100-110 <2 <15 <2 <2 Y1044W 100 100 NTg NTg NTg Y1044F 95-100 90-100 NTg NTg NTg Y1044C 90-95 <2 NTg NTg NTg Y1044A 100 <2 <5 <2 <2 Y401W/Y1044W 90-95 <2 NTg NTg NTg Y401F/Y1044F 90-95 95-100 NTg NTg NTg Y401C/Y1044C 100 <2 NTg NTg NTg Y401A/Y1044A 90-95 <2 <2 <2 <2 Y401F/Y1044W 100 100 NTg NTg NTg Y401C/Y1044W 100 <2 NTg NTg NTg Y401A/Y1044W 100-110 <2 NTg NTg NTg Y401W/Y1044F 100 100 NTg NTg NTg a The levels of cell surface expression, transport activity, ATP binding, nucleotide trapping, and ATP hydrolysis by the wild-type protein were taken to be 100%, and these activities in mutant Pgps were expressed relative to wild-type levels. Login to comment
118 Photoaffinity Labeling of Wild-Type and Y401A, Y1044A, Y401A/Y1044A, and Y401W Mutant Pgps with [125 I]IAAP. Login to comment
131 Binding of the fluorescent nucleotide analogue TNP-ATP was assessed by determining the increase in the fluorescence signal (excitation at 408 nm, emission at 540) of TNP-ATP (2.5-80 µM) associated with purified wild-type and mutant Pgps (Y401W, Y401A, Y1004A, and Y401A/Y1004A) incorporated into proteoliposomes as described previously (42). Login to comment
133 The concentration of TNP-ATP required for half-maximal binding was determined by measuring the signal of increasing concentrations of TNP-ATP in the presence of proteoliposomes (20-25 µg of protein/mL for the wild-type protein and Y401W mutant and 50-100 µg of protein/mL for Y401A, Y1044A, and Y401A/ Y1044A mutant proteins) and in the presence and absence of 10 mM ATP. Login to comment
144 We generated the single mutants Y401A, Y1044A, Y401C, Y1044C, Y401F, Y1044F, Y401W, Y1044W, and Y401L. Login to comment
145 We also generated the double mutants Y401A/Y1044A, Y401F/Y1044F, Y401W/Y1044W, Y401C/Y1044C, Y401A/Y1044W, Y401F/Y1044W, Y401C/ Y1044W, and Y401W/Y1044F. Login to comment
154 The transport function is abrogated in Y401A mutant Pgp (Figure 2A), but Y401F and Y401W show the same transport activity as wild-type Pgp (Figure 2B,C). Login to comment
159 Furthermore, it would be expected that the substitution of both Y401 and Y1044 with A and C (Y401A/Y1044A and Y401C/Y1044C) would abrogate function, whereas substitutions with F and W in both NBDs (Y401F/Y1044F and Y401W/Y1044W) would retain functionality. Login to comment
161 We also found loss of function in mutants Y401A/ Y1044W and Y401C/Y1044W. Login to comment
163 The cell surface expression of wild-type and mutant Pgps was assessed by staining Pgp in intact cells with human Pgp-specific monoclonal antibody MRK-16 (33) followed by flow cytometry as described in Experimental Procedures: (A) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y401A, (- - -) Y401C, (-‚-) Y401W, and (-‚‚-) Y401F, (B) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y1044A, (- - -) Y1044C, (-‚-) Y1044W, and (-‚‚-) Y1044F, and (C) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y401A/Y1044A, (- - -) Y401C/Y1044C, (-‚-) Y401W/ Y1044W, and (-‚‚-) Y401F/Y1044F. Login to comment
165 Calcein-AM efflux mediated by wild-type and mutant Pgps was monitored by flow cytometry as described in Experimental Procedures: (A) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y401A, (- - -) Y1044A, and (-‚‚-) Y401A/ Y1044A, (B) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y401F, (- - -) Y1044F, and (-‚‚-) Y401F/Y1044F, and (C) (thin line) pTM1, (thick line) wild type, (‚‚‚) Y401W, (- - -) Y1044W, and (-‚‚-) Y401W/Y1044W. Login to comment
169 Binding of 8-Azido[R-32 P]ATP to Wild-Type, Y401A, Y1044A, Y401A/Y1044A, Y401W, Y401C, and Y401L Mutant Pgps. Login to comment
173 However, incorporation of 8-azido[R-32 P]ATP was drastically attenuated in the Y401A, Y1044A, and Y401A/ 1044A mutant Pgps, compared to that in wild-type Pgp (Figure 3B). Login to comment
174 The level of 8-azido[R-32 P]ATP incorporated into Y401A, Y1044A, and Y401A/Y1044A equaled 10-15, 3-5, and ~1% (undetectable level), respectively. Login to comment
176 Trapping of 8-Azido[R-32 P]ADP into Wild-Type, Y401A, Y1044A, Y401A/Y1044A, and Y401W Mutant Pgps. Login to comment
179 In Figure 4A, we show that wild-type and Y401W Pgps trap equivalent levels of 8-azido[R-32 P]ADP, whereas in Y401A and Y1044A mutant Pgps, Vi-induced 8-azidoADP trapping was not detected even in the presence of 30 µM verapamil (Figure 4C). Login to comment
194 (C) Vi-induced nucleotide trapping was not observed with Y401A or Y1044A mutant Pgp. Login to comment
195 Wild-type and Y401A and Y1044A mutant Pgps were purified and reconstituted into proteoliposomes as described in Experimental Procedures. Login to comment
210 To examine whether the differences in transport function were due to an alteration in the binding properties of transport substrate resulting from the Y401A, Y1044A, Y401A/ Y1044A, and Y401W substitutions, we compared the photocross-linking of mutant Pgps with IAAP. Login to comment
212 Mutants Y401W, Y401A, Y1044A, and Y401A/Y1044A were all labeled with [125 I]IAAP at levels comparable to that of wild-type Pgp, and cyclosporine A inhibited IAAP incorporation in both wild-type and mutant Pgps (data not shown). Login to comment
214 ATPase ActiVities of Wild-Type, Y401A, Y1044A, Y401A/ Y1044A, and Y401W Pgps. Login to comment
218 However, ATPase activity of the Y401A, Y1044A, and Y401A/Y1044A mutants was comparable to that of control HighFive cells not expressing Pgp. Login to comment
243 Binding of TNP-ATP to Wild-Type, Y401A, Y1044A, Y401A/Y1044A, and Y401W Pgps. Login to comment
252 However, the concentration required for half-maximal binding of TNP-ATP to Y401A mutant Pgp was considerably higher than that for the wild type (105.5 µM, Figure 7C). Login to comment
253 Furthermore, the binding of TNP-ATP to Y1044A and Y401A/Y1044A mutant Pgs was not saturable up to 100 µM (Figure 7C,D), and for this reason, we could not determine the affinity of the fluorescent nucleotide for these mutants. Login to comment
254 It is worth noting that for experiments in panels C and D of Figure 7, 2-4 times more protein, Y401A, Y1044A, and Y401A/Y1004A mutant Pgps, was used to obtain signal above the liposomal FIGURE 7: Binding of TNP-ATP to proteoliposomes containing purified wild-type, Y401W, Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Login to comment
257 (B-D) The affinity of TNP-ATP for wild-type (b) and Y401W mutant Pgp (2) (20-25 µg of protein/mL), Y401A (9) and Y1044A (0) mutant Pgps (50-100 µg of protein/mL), double mutant Y401A/Y1044A Pgp (100 µg of protein/mL) (1), and liposomes (O) (dashed line in panels B-D) was determined by using increasing concentrations of TNP-ATP. Login to comment
262 It is clear that even in the case of the Y401A mutant, the apparent Kd value is significantly underestimated. Login to comment
268 [R-32 P]ATP Hydrolysis by Wild-Type, Y401A, Y1044A, and Y401A/Y1044A Mutant Pgps. Login to comment
272 Consistent with the results with crude membrane preparations, we found that ATP hydrolysis was completely abolished by substitution of tyrosine with alanine at position 401 and/or 1044 in proteoliposomes reconstituted with purified mutant proteins. Login to comment
293 On the other hand, the Y f A mutants do not show transport or ATP hydrolysis and the Y401A mutant exhibited significantly attenuated (<20%) 8-azido[R-32 P]ATP and negligible TNP-ATP binding (Table 1 and Figures 3B and 7C). Login to comment

PMID: 18058211 [PubMed] Sauna ZE et al: "Genomics and the mechanism of P-glycoprotein (ABCB1)."

No. Sentence Comment

79 However, transport was completely abolished in Y401A, Y1044A, and Y401/Y1044A mutant P-gps and there was no detectable binding of 8-azido[α-32 P]ATP or TNP-ATP, Vi-induced trapping of nucleotide, and ATP hydrolysis in these mutant P-gps. Login to comment

PMID: 21452853 [PubMed] Ohnuma S et al: "Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1) function by 5'-fluorosulfonylbenzoyl 5'-adenosine: evidence for an ATP analogue that interacts with both drug-substrate-and nucleotide-binding sites."

No. Sentence Comment

30 In addition, this FSBA interaction at the TMDs does not require functional ATP sites, as the IAAP labeling in the Y401A/Y1044A double mutant lacking ATP-binding capacity is still inhibited by FSBA. Login to comment
38 High-Five insect cells (Invitrogen, Carlsbad, CA) were infected with the baculovirus carrying the human MDR1 cDNA (either wild-type or Y401A/ 1044A or the cys-less mutant where all seven native cysteine residues are replaced with alanine) with a 6X histidine tag at the C-terminal end as described previously.33 Crude membranes were prepared and stored at À70 °C as described previously.33,34 Purification and Reconstitution of P-gp. Login to comment
156 Similar results were obtained when the mutant P-gp (Y401A/Y1044A), which lacks the capacity to bind nucleotides, was used in lieu of the wild-type protein (Figure 6D). Login to comment
188 (B) The apparent affinity of FSBA for the substrate-binding sites of the Y401A/Y1044A mutant-P-gp, which cannot bind ATP, was estimated as described in (A). Login to comment
190 Photoaffinity labeling of wild-type (C) and Y401A/1044A mutant (D) with IAAP after the formation of FSBA-P-gp adduct. Login to comment
257 The binding of the transport substrate IAAP to wild-type P-gp or mutant Y401A/1044A (that cannot bind to ATP) is not affected by the formation of an FSBA-P-gp adduct (Figure 6C,D), suggesting that FSBA is not cross-linked to the IAAP binding site. 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

52 Between the Walker A and B sequences is found a linker peptide with the sequence LSGGQ, also known as the C-region or ABC signature sequence, as it is the hallmark of Table 1 - Summary of mutational analysis of conserved residues in nucleotide-binding domains of Pgp Domain Source Residue number Function Reference NBD1 NBD2 A-loop Human Y401A Y1044A No ATP binding/hydrolysis Kim et al. (submitted for publication) Walker A Mouse K429N K1072N Normal ATP binding but no hydrolysis Azzaria et al. (1989) G431A G1073A Human C431 C1074 ATP protects from modification by N-ethylmaleimide Loo and Clarke (1995) Disulfide bond formation between Walker A domains of both NBDs Urbatsch et al. (2001) Human K433M K1076M Decreased ATP-binding Muller et al. (1996) No ATP hydrolysis Szakacs et al. (2000) No vanadate-trapping, but aluminum and beryllium fluoride-induced trapping normal Q-loop Mouse Q471 Q1114 Not essential for ATP hydrolysis but may be involved in communication with drug-substrate sites Urbatsch et al. (2000a) LSGGQ or linker peptide or signature motif Mouse S528A S1173A Normal ATP binding but no hydrolysis Tombline et al. (2004a) Human S532R Decreased cell surface expression Hoof et al. (1994) Human G534C G1179C No ATP hydrolysis Loo et al. (2002) Human G534D Decreased cell surface expression Hoof et al. (1994) No drug resistance Normal cell surface expression Bakos et al. (1997) No ATP hydrolysis Human G534D/V G1179D Interdomain communication Szakacs et al. (2001) Human Q535C Q1180C No ATP hydrolysis Loo et al. (2002) Human K536Q Decreased drug resistance Hoof et al. (1994) LSGGQ or linker peptide or signature motif Human K536R Increased colchicine resistance (normal ATP hydrolysis?) Login to comment