ABCMdb

ABCB1 p.Tyr1044Cys

Predicted by SNAP2:	A: D (91%), C: D (85%), D: D (95%), E: D (95%), F: D (85%), G: D (95%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (91%), W: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D,

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Publications
[hide] The conserved tyrosine residues 401 and 1044 in AT... Biochemistry. 2006 Jun 20;45(24):7605-16. 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 [PMID:16768456]

Abstract [show]
Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).

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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
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
265 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
297 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