ABCMdb

ABCB1 p.Tyr1044Trp

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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

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
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
157 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 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
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
166 Y1044W and Y401W/Y1044F were functional (Table 1). 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