ABCMdb

ABCB1 p.Tyr401Trp

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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
115 The labeled crude membranes containing wild-type and Y401W Pgps were treated with trypsin at a trypsin:protein ratio of 1:10 (w/w) for 5 min at 37 °C, and the reaction was terminated by adding a 5-fold excess of trypsin inhibitor. Login to comment
117 The same blot was used to obtain the 32 P signal by exposing it to an X-ray film at -70 °C. Colocalization of the two signals indicated that the 32 P-labeled radionucleotide was incorporated into both halves of wild-type and Y401W Pgps. 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
160 However, we found that Y401W/Y1044W double mutant Pgp exhibited a complete loss of function, measured as efflux of calcein-AM. 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
166 Y1044W and Y401W/Y1044F were functional (Table 1). 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
172 Quantification of the radiolabel signal shows that the amount of 8-azido[R-32 P]ATP incorporated in Y401W is approximately 80% of that in wild-type Pgp. 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
181 (A) Crude membranes prepared from HighFive insect cells overexpressing the wild type or the Y401W mutant Pgp were incubated with 10 µM (with 5-10 µCi/nmol) 8-azido[R-32P]ATP in the absence (-) or presence (+) of 10 mM ATP in the ATPase assay buffer as described in Experimental Procedures, photo-cross-linked, and separated on a 7% gel. Login to comment
188 (A) Vior BeFx-induced trapping of 8-azido- [R-32P]ADP in wild-type or Y401W mutant Pgp was monitored as described in Experimental Procedures. Login to comment
189 The autoradiogram depicts 8-azido[R-32P]ADP incorporated into the wild-type and Y401W mutant protein in the presence and absence of Vi or BeFx. Login to comment
190 (B) Distribution of trapped 8-azido[R-32P]ADP in the Nand C-ATP sites of wild-type and Y401W mutant Pgp. Login to comment
202 To determine whether the mutation in the Nor C-terminal NBD affects nucleotide trapping only at that site, the wild-type and Y401W Pgps were subjected to mild trypsin digestion following photocross-linking of trapped 8-azido[R-32 P]ADP in the presence of Vi as described in Experimental Procedures. Login to comment
204 An immunoblot of wild-type and Y401W Pgps after mild trypsinization was performed with polyclonal antibody PEPG-13 to detect the N-half of Pgp and Pgp-specific monoclonal antibody C219, which recognizes both halves of Pgp (34). Login to comment
205 Mild trypsinization of wild-type and Y401W Pgps showed that both the Nand C-terminal halves trap the radionucleotide (Figure 4B) to similar levels, indicating that the Y401W mutation does not affect the random selection of NBDs in initiating the catalytic cycle (42). Login to comment
207 To determine whether the trapped nucleotide is 8-azidoATP or 8-azidoADP, we incubated the wild-type and Y401W mutant Pgps in the presence of Vi with either 8-azido[R-32 P]ATP or 8-azido[γ-32 P]ATP (Figure 5). Login to comment
209 We found that in both the wild type and the Y401W mutant there was no 32 P signal associated with the Pgp band when 8-azido[γ-32 P]ATP was used, suggesting that the trapped moiety is the nucleotide diphosphate, and this is indeed the posthydrolysis transition state. 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
217 As shown in Figure 6, the wild-type Pgp as well as the Y401W mutant exhibited Vi-sensitive ATPase activity that was stimulated by verapamil. Login to comment
220 Other drug substrates such as vinblastine and valinomycin also gave comparable results, stimulating ATP hydrolysis only in the wild-type Pgp and the Y401W mutant (data not given). Login to comment
222 Although it is clear that mutant Y401W can hydrolyze ATP, the level of activity when 5 mM ATP is used is in the range of 60-75%, compared to that of wild-type protein. Login to comment
224 Thus, we determined the Km(ATP) for wild-type and mutant Y401W Pgps both in the presence and in the absence of 30 µM verapamil. Login to comment
226 The kinetic parameters of ATP hydrolysis by wild-type and Y401W mutant Pgp derived from the data in Figure S1 of the Supporting Information indicate that the maximal velocity FIGURE 5: Vi-induced trapping of nucleotide in wild-type and Y401W mutant Pgps by incubating with 8-azido[R-32P]ATP or 8-azido[γ-32P]ATP. Login to comment
227 Vi-induced trapping was carried out with wild-type or mutant Y401W Pgp (0.5 mg of protein/mL) in ATPase assay buffer (see Experimental Procedures) using either 8-azido- [R-32P]ATP (50 µM with 5 µCi/nmol) or 8-azido[γ-32P]ATP (50 µM with 5 µCi/nmol) as the nucleotide. Login to comment
237 of ATP hydrolysis by both the Y401W mutant and the wild-type protein is in the same range. Login to comment
240 These results suggest that though there is a small decrease in the affinity for ATP in the Y401W mutant, this substitution is tolerated by Pgp and unlikely to have any effect under physiological conditions. Login to comment
241 This conclusion is strengthened by the findings that the Vmax values for wild-type and mutant Y401W Pgps are comparable. Login to comment
242 This is also consistent with the similar transport activities in both wild-type and Y401W mutant Pgps (Figure 2 and Table 1). Login to comment
243 Binding of TNP-ATP to Wild-Type, Y401A, Y1044A, Y401A/Y1044A, and Y401W Pgps. Login to comment
250 The increase in fluorescence upon binding of TNP-ATP to wild-type and Y401W mutant Pgps was concentration-dependent and saturable (Figure 7B), and the apparent Kd(TNP-ATP) for the mutant Pgp, Y401W, was 32.8 µM compared to a value of 11.2 µM for wild-type Pgp. Login to comment
251 Thus, though mutant Y401W hydrolyzes ATP and retains transport function, the affinity for ATP is reduced 2-2.5-fold. 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
292 Y401W mutant Pgp in the reconstituted system exhibits properties similar to those of the wild-type protein except for a somewhat reduced affinity for ATP. Login to comment

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

No. Sentence Comment

77 Although the mutants Y401F, Y401W, Y1044F and Y1044W transported fluorescent substrates similar to wild-type protein, there was a significant decrease (2-2.5 fold) in the affinity of the nucleotide for the Y401W mutant P-gp. Login to comment