ABCMdb

ABCB1 p.Asp177Cys

None has been submitted yet.

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
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
115 Cross-linking of ICL1 (TMD1) and ICL3 (TMD2) in Close Proximity Also Activates P-gp ATPase Activity-To test if cross-linking of other segments predicted to undergo large conformational changes during the reaction cycle would also activate P-gp ATPase activity, we performed cross-linking studies on mutant D177C/N820C. Login to comment
120 Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M. Login to comment
129 A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). Login to comment
132 C, membranes expressing mutants D177C/N820C or L175C/N820C 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
137 Prior to cross-linking, mutant D177C/N820C showed verapamil-stimulated ATPase activity that was similar to the Cys-less parent. Login to comment
138 The basal ATPase activity of the M4M cross-linked D177C/N820C mutant was similar to that of uncross-linked mutant assayed in the presence of verapamil (Fig. 3B). Login to comment
139 No further stimulation of ATPase activity was observed when the M4M cross-linked D177C/N820C was assayed in the presence of verapamil. Login to comment
143 In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 °C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment
144 Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment
145 One possibility is that a separate M1M molecule preferentially modifies each cysteine of mutant D177C/ N820C as it was found that cross-linking was reduced when the concentration of M1M was increased (data not shown). Login to comment
146 We previously observed that mutant L175C/N820 only showed efficient cross-linking with a narrow concentration range of M1M (about 10-20 ␮M). Login to comment
150 We tested the effects of M4M and M17M cross-linking on the ATPase activity of mutant L175C/N820C and found that the effects were similar to those observed with mutant D177C/ N820C (Fig. 3C). 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
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
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
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
113 Cross-linking of ICL1 (TMD1) and ICL3 (TMD2) in Close Proximity Also Activates P-gp ATPase Activity-To test if cross-linking of other segments predicted to undergo large conformational changes during the reaction cycle would also activate P-gp ATPase activity, we performed cross-linking studies on mutant D177C/N820C. Login to comment
117 Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M. Login to comment
126 A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). 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
134 Prior to cross-linking, mutant D177C/N820C showed verapamil-stimulated ATPase activity that was similar to the Cys-less parent. Login to comment
135 The basal ATPase activity of the M4M cross-linked D177C/N820C mutant was similar to that of uncross-linked mutant assayed in the presence of verapamil (Fig. 3B). Login to comment
140 In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 &#b0;C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment
141 Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment
142 One possibility is that a separate M1M molecule preferentially modifies each cysteine of mutant D177C/ N820C as it was found that cross-linking was reduced when the concentration of M1M was increased (data not shown). 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
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: 20394729 [PubMed] Loo TW et al: "Human P-glycoprotein is active when the two halves are clamped together in the closed conformation."

No. Sentence Comment

97 Verapamil and rhodamine B were selected for study because thiol-reactive derivatives of these drug substrates have been used extensively in cysteine mutagenesis studies to characterize the X-link 170 kDa + + + _ + ++ + + + T176C D177C D178C L175C T176C D177C D178C L175C N820C + D821C + M1M A822C + ++ + T176C D177C D178C L175C B 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) X-link 170 kDa 0 2.2 C 0 20 40 60 80 100 PercentCross-linked 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) 0 2.2 A Fig. 2. Login to comment

PMID: 24275649 [PubMed] Loo TW et al: "Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein."

No. Sentence Comment

114 Cross-linking of IH2 and IH3 Inhibits Activity-In a previous cysteine mutagenesis and cross-linking study, we showed that clamping ICL3 (N820C) in close proximity to ICL1 (D175C or D177C) by cross-linking cysteines with short cross-linkers activated P-gp ATPase activity (15, 33). Login to comment