ABCMdb

PMID: 16768456

Kim IW, Peng XH, Sauna ZE, FitzGerald PC, Xia D, Muller M, Nandigama K, Ambudkar SV
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.
Biochemistry. 2006 Jun 20;45(24):7605-16., 2006-06-20 [PubMed]

Sentences

No. Mutations Sentence Comment

86 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys ABCB1 p.Tyr1044Cys ABCB1 p.Tyr1044Cys ABCB1 p.Tyr1044Cys ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala Photoaffinity Labeling of Wild-Type and Y401A, Y1044A, Y401A/Y1044A, and Y401W Mutant Pgps with [125 I]IAAP. Login to comment 131 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys ABCB1 p.Tyr401Phe We generated the single mutants Y401A, Y1044A, Y401C, Y1044C, Y401F, Y1044F, Y401W, Y1044W, and Y401L. Login to comment 145 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe 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 153 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys Figure 2 illustrates that in a flow cytometry assay HeLa cells expressing wild-type Pgp show a reduced level of accumulation of fluorescent calcein, whereas cells expressing equivalent amounts of Y401L and Y401C mutant Pgps show partial (30-50%) transport function (Table 1). Login to comment 154 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Phe 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 157 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys Transport of calcein-AM is abolished in mutants Y1044A and Y1044C (Figure 2A and Table 1), whereas the Y1044F and Y1044W mutants exhibited transport activity comparable to that of wild-type Pgp (Figure 2B,C). Login to comment 159 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys ABCB1 p.Tyr401Phe 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Trp However, we found that Y401W/Y1044W double mutant Pgp exhibited a complete loss of function, measured as efflux of calcein-AM. Login to comment 161 ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp We also found loss of function in mutants Y401A/ Y1044W and Y401C/Y1044W. Login to comment 162 ABCB1 p.Tyr401Phe However, mutants Y401F/ FIGURE 1: Expression of wild-type and mutant Pgps expressed in Vaccinia virus-infected HeLa cells. Login to comment 163 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Cys ABCB1 p.Tyr1044Cys ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe ABCB1 p.Tyr1044Phe ABCB1 p.Tyr401Phe ABCB1 p.Tyr401Phe 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Trp ABCB1 p.Tyr1044Phe Y1044W and Y401W/Y1044F were functional (Table 1). Login to comment 169 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp 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 173 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 175 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys In Y401L and Y401C mutants, 30-50% of the azidonucleotide was incorporated. Login to comment 176 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala Trapping of 8-Azido[R-32 P]ADP into Wild-Type, Y401A, Y1044A, Y401A/Y1044A, and Y401W Mutant Pgps. Login to comment 179 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp (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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp (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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp (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 194 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala (C) Vi-induced nucleotide trapping was not observed with Y401A or Y1044A mutant Pgp. Login to comment 195 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala Wild-type and Y401A and Y1044A mutant Pgps were purified and reconstituted into proteoliposomes as described in Experimental Procedures. Login to comment 196 ABCB1 p.Tyr1044Ala Proteoliposomes containing wild-type (20 µg of protein/mL) or Y401 and Y1044A mutant Pgps (150 µg of protein/mL) were incubated at 37 °C with 50 µM 8-azido[R-32P]ATP in the presence of 50 µM verapamil and in the presence and absence of 0.3 mM Vi for 10 min. Login to comment 200 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys cross-linking with 8-azidoATP at 4 °C, both Y401L and Y401C trap ~20% nucleotide in the presence of vanadate (Table 1). Login to comment 202 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ATPase ActiVities of Wild-Type, Y401A, Y1044A, Y401A/ Y1044A, and Y401W Pgps. Login to comment 217 ABCB1 p.Tyr401Trp 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 218 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 220 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp of ATP hydrolysis by both the Y401W mutant and the wild-type protein is in the same range. Login to comment 240 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp This conclusion is strengthened by the findings that the Vmax values for wild-type and mutant Y401W Pgps are comparable. Login to comment 242 ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala Binding of TNP-ATP to Wild-Type, Y401A, Y1044A, Y401A/Y1044A, and Y401W Pgps. Login to comment 250 ABCB1 p.Tyr401Trp ABCB1 p.Tyr401Trp 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 ABCB1 p.Tyr401Trp Thus, though mutant Y401W hydrolyzes ATP and retains transport function, the affinity for ATP is reduced 2-2.5-fold. Login to comment 252 ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala 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 ABCB1 p.Tyr401Trp ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala (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 ABCB1 p.Tyr401Ala It is clear that even in the case of the Y401A mutant, the apparent Kd value is significantly underestimated. Login to comment 264 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys Similar results were also obtained with Y401L and Y401C mutants (see Table 1). Login to comment 265 ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Cys The partial activity is observed only with Y401C in NBD1 and not with Y1044C in NBD2, suggesting that there might be cooperativity and slight asymmetry in NBD1 and NBD2. Login to comment 268 ABCB1 p.Tyr1044Ala ABCB1 p.Tyr1044Ala ABCB1 p.Tyr401Ala ABCB1 p.Tyr401Ala [R-32 P]ATP Hydrolysis by Wild-Type, Y401A, Y1044A, and Y401A/Y1044A Mutant Pgps. Login to comment 272 ABCB1 p.Tyr401Ala 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 292 ABCB1 p.Tyr401Trp 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 293 ABCB1 p.Tyr401Ala 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 294 ABCB1 p.Tyr1044Ala However, Y1044A mutant Pgp does not exhibit nucleotide (TNP-ATP and 8-azido[R-32 P]ATP) binding (Figures 3B and 7C). Login to comment 297 ABCB1 p.Tyr401Leu ABCB1 p.Tyr401Cys ABCB1 p.Tyr401Cys ABCB1 p.Tyr1044Cys ABCB1 p.Tyr1044Cys Consistent with this view is the fact that while mutants Y401L and Y401C are partially (30-50%) functional, mutants Y1044C and Y401C/Y1044C are completely nonfunctional (Table 1). Login to comment 308 ABCB1 p.Tyr401Cys On the other hand, in the Y401C mutant, the S group of the C residue interacts with C6 of the adenine base through hydrogen bonding (distance of 3.3 Å). Login to comment