ABCMdb

ABCB1 p.Met69Cys

Predicted by SNAP2:	A: N (78%), C: N (87%), D: D (53%), E: D (53%), F: N (82%), G: N (66%), H: N (61%), I: N (87%), K: D (53%), L: N (87%), N: N (78%), P: D (53%), Q: N (61%), R: D (53%), S: N (82%), T: N (82%), V: N (82%), W: D (53%), Y: N (72%),
Predicted by PROVEAN:	A: D, C: N, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] ATP hydrolysis promotes interactions between the e... Biochemistry. 2005 Aug 2;44(30):10250-8. Loo TW, Bartlett MC, Clarke DM
ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein.
Biochemistry. 2005 Aug 2;44(30):10250-8., 2005-08-02 [PMID:16042402]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.

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97 Figure 2A shows that the slower migrating product in mutants M68C(TM1)/Y950C(TM11), M68C- (TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C- (TM1)/A954C(TM11), and M69C(TM1)/F957C(TM11) was greatly reduced upon exposure to 10 mM dithiothreitol. Login to comment
105 Mutants M68C(TM1)/ Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), and M69C- (TM1)/F957C(TM11) all showed a time-dependent increase FIGURE 1: Model of packing of the TM segments of P-gp and location of the introduced cysteines. Login to comment
134 Membranes were prepared from HEK 293 cells (A), expressing mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C- (TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), M69C(TM1)/ F957C(TM11), or the negative mutant F72C(TM1)/F957C(TM11); (B) the single cysteine mutants M68C(TM1), M69C(TM1), Y950C(TM11), Y953C(TM11), A954C(TM11), or F957C(TM11); or (C) coexpressing the single cysteine mutants M68C(TM1) plus Y950C(TM11), M68C(TM1) plus Y953C(TM11), M68C(TM1) plus A954C(TM11), M69C(TM1) plus A954C(TM11), or M69C- (TM1) plus F957C(TM11). Login to comment
163 Accordingly, the histidine-tagged P-gp mutants M68C(TM1)/ Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), and M69C- (TM1)/F957C(TM11) and the Cys-less P-gp were expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment