ABCMdb

ABCB1 p.Phe1086Ala

Predicted by SNAP2:	A: D (75%), C: D (59%), D: D (91%), E: D (85%), G: D (80%), H: D (80%), I: D (71%), K: D (91%), L: D (59%), M: D (59%), N: D (80%), P: D (85%), Q: D (71%), R: D (85%), S: D (71%), T: D (75%), V: D (71%), W: D (59%), Y: N (53%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: 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,

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Publications
[hide] Human P-glycoprotein contains a greasy ball-and-so... J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.
J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3., [PMID:23733192]

Abstract [show]
The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.

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Sentences [show]
No. Sentence Comment
76 Effect of F1086A on Vanadate Trapping-The double cysteine mutant L443C/S909C was used to test whether the F1086A mutation inhibited vanadate trapping of nucleotide. Login to comment
78 Membranes prepared from cells expressing L443C/S909C or L443C/S909C/F1086A were incubated in the presence or absence of 5 mM MgATP plus 0.2 mM vanadate for 5 min at 37 &#b0;C. Samples were then treated with 1 mM copper phenanthroline for 15 min at 0 &#b0;C. The reactions were performed using a protein concentration of 0.4 mg/ml. Login to comment
83 The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells. Login to comment
106 The F1086A NBD1 Mutation Blocks ATP-dependent Extracellular Conformation Changes between the TMDs-Models of P-gp in the open and closed conformations are shown (Fig. 3, A and B). Login to comment
126 To test whether replacement of Phe-1086 with a small amino acid would affect maturation, mutants F1086A or Y1087A in the wild-type background were constructed and expressed in HEK 293 cells. Login to comment
127 Fig. 3D shows that the Y1087A but not the F1086A mutation inhibited P-gp maturation (Fig. 3D). Login to comment
128 Although the F1086A mutation did not inhibit folding, it still inhibited verapamil-stimulated ATPase activity (Fig. 3C). Login to comment
130 To address this question, we first tested whether the F1086A mutation blocked interactions with ATP or verapamil. Login to comment
138 A and B, structures of P-gp in the open (A) (17) or closed (B) (24) conformations are shown. C, histidine-tagged Cys-less P-gp or mutants A266C/F1086C, A266C, and F1086C (in Cys-less background) as well as wild-type P-gp and mutant F1086A ( in wild-type background) were isolated, and ATPase activities were measured in the presence of verapamil. Login to comment
141 E, membranes prepared from cells expressing L443C/S909C af9; F1086A were first treated with (af9;VO4) or without (afa;VO4) MgATP plus vanadate. Login to comment
146 To test whether mutant F1086A retained the ability to bind and hydrolyze ATP, we tested whether vanadate trapping of ATP would still inhibit cross-linking of mutant F1086A/L443C/S909C. Login to comment
151 Accordingly, membranes prepared from cells expressing mutant F1086A/ L443C(NBD1)/S909C(IH4) were preincubated with or without ATP plus vanadate for 5 min at 37 &#b0;C. Samples were then cooled on ice, treated with copper phenanthroline, and subjected to immunoblot analysis. Login to comment
152 Fig. 3E shows that vanadate trapping of nucleotide blocked cross-linking in both mutants L443C(NBD1)/S909C(IH4) and L443C(NBD1)/S909C(IH4)/ F1086A. Login to comment
153 These results show that the F1086A mutation did not inhibit ATP hydrolysis. Login to comment
154 A drug rescue assay was then used to test whether the F1086A mutation disrupted verapamil interactions with the TMDs. Login to comment
157 Accordingly, the F1086A mutation was introduced into the P709A processing mutant. Login to comment
159 Expression of mutant P709A/ F1086A yielded the 150-kDa immature form of P-gp as the major product (Fig. 3F). Login to comment
161 It is possible that the F1086A mutation inhibited activity by disrupting coupling between the NBDs and TMDs. Login to comment
162 Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling. Login to comment
170 To determine whether the F1086A mutation affected NBD/ TMD coupling, it was introduced into mutant T333C/L975C and subjected to cross-linking. Login to comment
171 It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B). Login to comment
172 These results suggest that F1086A disrupted NBD/TMD coupling. Login to comment
173 Replacement of Phe-1086 with Bulky Hydrophobic Amino Acids Yields Active Mutants-Mutant F1086A was inactive (Fig. 3C). Login to comment
182 Replacement of Ala-266 with Hydrophobic Residues Restores Activity of Mutant F1086A-Models (Fig. 3, A and B) and cross-linking results (21) predict that Ala-266 in IH2 is close to Phe-1086 in NBD2. Login to comment
183 Because a large hydrophobic amino acid might be required at the Ala-266/Phe-1086 interface for activity, we tested whether replacement of Ala-266 with the aromatic amino acids Tyr, Phe, or Trp could act as suppressor mutations to restore activity of the F1086A. Login to comment
184 We also predicted that replacement of Ala-266 with a small charged amino acid (Asp) would not restore activity to F1086A. Login to comment
185 It was found that replacement of Ala-266 with aromatic amino acids (A266Y, A266F or A266W) in the F1086A background yielded mature 170-kDa FIGURE 4. Login to comment
186 F1086A inhibits ATP-dependent cross-linking at the extracellular surface. Login to comment
187 A and B, membranes prepared from cells expressing mutant T333C/L975C (A) or T333C/L975C/F1086A (B) were treated with BMOE in the absence (None) or presence of nucleotides (ATP, AMP-PNP, or ADP). Login to comment
190 By contrast, mutant A266D/F1086A did not mature, although it could be rescued when expressed in the presence of cyclosporine A (Fig. 6B). Login to comment
192 Mutant A266D/ F1086A was first expressed in the presence of cyclosporine A to promote maturation before isolating the histidine-tagged protein. Login to comment
193 Fig. 6C shows that the verapamil-stimulated ATPase activities of mutants A266Y/F1086A, A266F/F1086A, and A266W/ F1086A were similar to that of wild-type P-gp. Login to comment
194 Mutant A266D/ F1086A showed little activity (Fig. 6C). Login to comment
195 The results show that the aromatic replacements to Ala-266 acted as second-site suppressor mutations to restore activity of F1086A. Login to comment
196 Replacement of Ala-266 with an aromatic amino acid might rescue defective TMD/NBD coupling and thereby restore activity to F1086A. Login to comment
197 Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs. Login to comment
216 Replacement of Ala-266 with aromatic residues restores F1086A activity. Login to comment
217 A, wild-type P-gp or A266X mutants containing the F1086A mutation were expressed in the absence of drug substrates. B, mutant A266D/F1086A was expressed in the presence (af9;) or absence (afa;) of cyclosporine A (Cyclo). Login to comment
219 The positions of mature (170 kDa) and immature (150 kDa) P-gps are indicated. C, ATPase activities of wild-type P-gp and F1086A mutants containing replacements to Ala-266 were measured in the presence of verapamil. Login to comment
221 D, membranes prepared from cells expressing mutants T333C/L975C (None), T333C/L975C/F1086A, or T333C/L975C/F1086A/A266F were treated with BMOE in the presence (af9;) or absence (afa;) of ATP. Login to comment
239 In addition, activity of the F1086A mutant could be restored if Ala-266 was replaced with an aromatic amino acid. 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.

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Sentences [show]
No. Sentence Comment
201 Although we showed previously that mutation of the adjacent Phe1086 to Ala also inhibited activity (16), the activity of P-gp was much more sensitive to changes to Tyr1087 . Login to comment

[hide] The Transmission Interfaces Contribute Asymmetrica... J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18. Loo TW, Clarke DM
The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.
J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18., [PMID:25987565]

Abstract [show]
P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 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. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.

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Sentences [show]
No. Sentence Comment
170 For example, the F1086A mutation abolished activity. Login to comment
171 Activity of the F1086A could be restored if the opposing Ala-266 residue in IH2 was replaced with an aromatic residue (23). Login to comment
178 Mutants F1086A, F1086L, and F1086W yielded mature 170 kDa protein as the major product, whereas F1086R yielded little mature P-gp (b0d;5%) (Fig. 4A). Login to comment
185 While the F1086A and F1086R mutations FIGURE 3. Login to comment