ABCMdb

ABCB1 p.Pro517Cys

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Publications

PMID: 22700974 [PubMed] Loo TW et al: "The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together."

No. Sentence Comment

10 It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. Login to comment
93 To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively. Login to comment
95 In the open conformation P517C and I1050C are predicted to be far apart (22.9 Å; measured from the ␣ carbons) but close (7.8 Å) in the closed conformation. Login to comment
96 The P517C/I1050C mutant was expressed in HEK 293 cells. Login to comment
99 To test the effect of covalently linking NBD1 and NBD2 on catalytic activity, ATPase activity of mutant P517C/NI1050C was measured in the presence or absence of verapamil before and after cross-linking with the short M4M cross-linker. Login to comment
106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
107 Linkage of the NBDs Activates P-gp ATPase Activity 26808 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 32•AUGUST 3, activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 ϩ 0.2 ␮mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 ϩ 0.3 ␮mol Pi/min/mg P-gp). Login to comment
108 Cross-linking of P517C/I1050C with M4M increased its basal ATPase activity (14-fold higher than the basal ATPase activity of untreated P-gp) (Fig. 2B). Login to comment
110 The enhanced activity of M4M cross-linked P517C/I1050C was not due to carry over of M4M during purification because M4M had little effect on Cys-less P-gp ATPase activity (Fig. 2B). Login to comment
111 Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B). Login to comment
112 The large increase in basal ATPase activity observed when mutant P517C/I1050C was cross-linked with the short M4M cross-linker appeared to be caused by trapping of the NBDs in close proximity rather than nonspecific cross-linking effects because treatment with the long M17M cross-linker caused a much smaller increase (about 2-fold) in basal ATPase activity (Fig. 2B). Login to comment
114 The cross-linking results observed with mutant P517C/ I1050C suggest that covalently linking NBD1 and NBD2 in close proximity with M4M mimics activation of ATPase activity with compounds that highly activate P-gp ATPase activity. Login to comment
120 Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M. Login to comment
123 A, membranes prepared from HEK 293 cells expressing the NBD1/ NBD2 mutant P517C/I1050C were treated without (Control) or with cross-linkers M4M or M17M. Login to comment
125 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment
136 It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B). Login to comment
158 In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp. Login to comment
163 These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation. Login to comment
194 Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking. Login to comment
225 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment
226 D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (-) or presence (ϩ) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis. Login to comment
232 An explanation for the increased activity is that P517C and I1050C are located at the N-terminal end and central segments of the NBDs close to the LSGGQ and Walker A sites of NBD1 and NBD2, respectively. Login to comment
233 Cross-linking P517C/I1050C (or D177C/ N820C) with M4M would hold the Walker A and LSGGQ sites in close proximity (Fig. 7A) to increase the probability of generating the ATP-bound sandwich conformation and therefore increase the basal ATPase activity. Login to comment
236 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment
264 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment
91 To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively. Login to comment
94 The P517C/I1050C mutant was expressed in HEK 293 cells. Login to comment
97 To test the effect of covalently linking NBD1 and NBD2 on catalytic activity, ATPase activity of mutant P517C/NI1050C was measured in the presence or absence of verapamil before and after cross-linking with the short M4M cross-linker. Login to comment
104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
105 Linkage of the NBDs Activates P-gp ATPase Activity 26808 activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 af9; 0.2 òe;mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 af9; 0.3 òe;mol Pi/min/mg P-gp). Login to comment
109 Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B). Login to comment
122 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment
133 It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B). Login to comment
189 Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking. Login to comment
219 D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (afa;) or presence (af9;) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis. Login to comment
229 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment
257 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment

PMID: 25053414 [PubMed] Loo TW et al: "Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity."

No. Sentence Comment

258 Cysteines L175C/N820C are located in the intracellular loops (Fig. 1, C and D), whereas cysteines P517C/I1050C are located in the NBDs (Fig. 1, C and D). Login to comment
259 An explanation for the difference is that linkage of the homologous halves at the L175C/N820C or P517C/I1050C sites would tend to favor the closed conformation (Fig. 1C) to FIGURE 9. Login to comment

PMID: 25600711 [PubMed] Pan L et al: "Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics."

No. Sentence Comment

203 We did not simulate cross-linking per se, but our structure showed direct side-chain contact between the crosslinked pair LSGGQ (P517C) and Walker A (I1050C) (mouse P513-I1046) showing agreement between our homology model and the biochemical data. Login to comment

PMID: 26507655 [PubMed] Loo TW et al: "Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein."

No. Sentence Comment

67 Mutant P517C/I1050C was cross-linked in membranes at 0 &#b0;C with 50 òe;M 1,4-butanediyl bismethanethiosulfonate (BMTS) in the absence or presence of 1 òe;M tariquidar (30). Login to comment
118 The conditions for cross-linking mutants T333C/L975C and P517C/I1050C, however, were different. Login to comment
120 Cross-linking of mutant P517C/I1050C was performed on membranes using BMTS cross-linker (30). Login to comment