ABCMdb

ABCB1 p.Asp177Cys

Predicted by SNAP2:	A: D (63%), C: D (71%), E: N (66%), F: D (80%), G: D (71%), H: D (80%), I: D (80%), K: D (80%), L: D (80%), M: D (80%), N: N (53%), P: D (85%), Q: D (71%), R: D (80%), S: N (61%), T: D (63%), V: D (75%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, E: N, 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, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] The ATPase activity of the P-glycoprotein drug pum... J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14. Loo TW, Bartlett MC, Detty MR, Clarke DM
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.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PMID:22700974]

Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 A) or long (22 A) cross-linkers to identify domain-domain interactions that activate ATPase activity. 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. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Human P-glycoprotein is active when the two halves... Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.
Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13., [PMID:20394729]

Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 A and 20 A apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 A) at 0 degrees C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Locking intracellular helices 2 and 3 together ina... J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25. Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PMID:24275649]

Abstract [show]
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(beta,gamma-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.

Comments [show]

None has been submitted yet.

Sentences [show]
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