ABCMdb

PMID: 23733192

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., [PubMed]

Sentences

No. Mutations Sentence Comment

56 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys IH2 appears to be particularly important because we showed that a cysteine introduced into IH2 (A266C) could be directly cross-linked to a cysteine in NBD2 (F1086C) but the mutant was inactive (21). Login to comment 62 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys Because mutant A266C/F1086C was inactive, we characterized the Ala-266/ Phe-1086 interface to determine its role in the transport cycle. Login to comment 76 ABCB1 p.Leu443Cys ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala 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 77 ABCB1 p.Leu443Cys Vanadate trapping inhibits cross-linking of L443C/S909C P-gp (20). Login to comment 78 ABCB1 p.Leu443Cys ABCB1 p.Leu443Cys ABCB1 p.Phe1086Ala 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 82 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20327 Effect of Mutations on ATP-dependent Cross-linking of P-gp Extracellular Segments-Mutant T333C/L975C contains cysteines predicted to reside at the extracellular ends of TM segments 6 and 12, respectively (30). Login to comment 83 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala ABCB1 p.Ala266Phe The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells. Login to comment 90 ABCB1 p.Ala259Gln To test whether IH2 was important for folding, the effect of mutations to residues Ala-259-Gln-270 (IH2 and flanking residues) on maturation was examined. Login to comment 96 ABCB1 p.Gly269Val ABCB1 p.Gly268Val ABCB1 p.Val264Ser ABCB1 p.Phe267Ala The I261S, V264S, F267A, G268V, and G269V mutations blocked maturation such that the 150-kDa immature protein was the major product (Fig. 1B). Login to comment 97 ABCB1 p.Ala259Leu ABCB1 p.Ala266Leu Mutants A259L and A266L were similar to wild-type P-gp as the 170-kDa mature protein was by far the most prominent product. Login to comment 98 ABCB1 p.Ala260Leu ABCB1 p.Thr263Ala ABCB1 p.Ile265Ser ABCB1 p.Arg262Ala Mutants A260L, R262A, T263A, I265S, and Q270A yielded a mixture of mature and immature P-gp (Fig. 1, B and C). Login to comment 100 ABCB1 p.Gln158Gly In contrast to IH2, we found that none of the mutations introduced to IH1 and flanking residues (Gln-158-Gly-169) substantially inhibited maturation of P-gp (Fig. 2B). Login to comment 102 ABCB1 p.Asp164Ala ABCB1 p.Gly161Val Mutants I160A, G161V, and D164A were selected for analysis because the recent high-resolution crystal structure of C. elegans P-gp showed that residues at these positions interacted with NBD1 (17). Login to comment 106 ABCB1 p.Phe1086Ala 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 109 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys In a previous study, we provided biochemical evidence that Ala-266 was indeed close to Phe-1086 in NBD2 because mutant A266C/F1086C showed robust cross-linking even when treated with oxidant at 0 &#b0;C to slow molecular motion (21). Login to comment 110 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Ala266Phe The Ala-266/Phe-1086 contact point appeared to be critical for function because mutant A266C/F1086C was inactive (21). Login to comment 111 ABCB1 p.Leu443Cys By contrast, introduction of cysteine mutations at the equivalent sites between IH4 (S909C) and NBD1 (L443C) (Fig. 3, A and B) had no effect on activity (21). Login to comment 117 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump 20328 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 28ߦJULY 12, 2013 To determine whether one or both cysteine mutations in mutant A266C/F1086C inhibited activity, mutants were constructed in a Cys-less background that contained only Cys-266 or Cys-1086. Login to comment 119 ABCB1 p.Phe1086Cys It was observed that only the F1086C mutation caused a drastic reduction in verapamil-stimulated ATPase activity (Fig. 3C). Login to comment 120 ABCB1 p.Phe1086Cys An explanation for the ability of the F1086C mutation to inhibit P-gp activity was that the mutation affected folding of P-gp. Login to comment 121 ABCB1 p.Phe1086Cys ABCB1 p.Phe1086Cys ABCB1 p.Tyr1087Cys To test whether the F1086C mutation inhibited folding, the F1086C mutant and the Y1087C mutant were expressed in the absence of drug substrates. Login to comment 122 ABCB1 p.Tyr1087Cys The Y1087C mutant was included because an aromatic residue at position 1087 is highly conserved in ABC proteins (17). Login to comment 123 ABCB1 p.Phe1086Cys ABCB1 p.Tyr1087Cys It was found that both the F1086C and the Y1087C mutations inhibited maturation of Cys-less P-gp (Fig. 3D). Login to comment 125 ABCB1 p.Phe1086Cys In addition, it was possible that replacement of Phe-1086 with a cysteine caused a defect in the structure of P-gp that would be different if it had been replaced with a smaller amino acid. Login to comment 126 ABCB1 p.Phe1086Ala ABCB1 p.Tyr1087Ala 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 ABCB1 p.Phe1086Ala ABCB1 p.Tyr1087Ala Fig. 3D shows that the Y1087A but not the F1086A mutation inhibited P-gp maturation (Fig. 3D). Login to comment 128 ABCB1 p.Phe1086Ala Although the F1086A mutation did not inhibit folding, it still inhibited verapamil-stimulated ATPase activity (Fig. 3C). Login to comment 130 ABCB1 p.Phe1086Ala To address this question, we first tested whether the F1086A mutation blocked interactions with ATP or verapamil. Login to comment 138 ABCB1 p.Ala266Cys ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Phe1086Cys ABCB1 p.Phe1086Ala 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 ABCB1 p.Leu443Cys ABCB1 p.Phe1086Ala 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 144 ABCB1 p.Pro709Ala F, the P709A processing mutation wasintroducedintomutantF1086A.Mutantswereexpressedinthepresence (af9;) or absence (afa;) of verapamil (Ver), and whole cell extracts were subjected to immunoblot analysis. Login to comment 145 ABCB1 p.Ser1077Gly IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20329 It is possible that mutations to Phe-1086 could affect ATP interactions because the residue is close to the Walker A site in NBD2 (residues Ser-1077 to Gly-1084). Login to comment 146 ABCB1 p.Leu443Cys ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala These results show that the F1086A mutation did not inhibit ATP hydrolysis. Login to comment 154 ABCB1 p.Phe1086Ala A drug rescue assay was then used to test whether the F1086A mutation disrupted verapamil interactions with the TMDs. Login to comment 157 ABCB1 p.Pro709Ala ABCB1 p.Phe1086Ala Accordingly, the F1086A mutation was introduced into the P709A processing mutant. Login to comment 158 ABCB1 p.Pro709Ala The P709A mutant was selected because the mutation is located in the linker region that connects the two halves of P-gp and is outside the ATP-and drug-binding sites (Fig. 3, A and B). Login to comment 159 ABCB1 p.Pro709Ala ABCB1 p.Phe1086Ala Expression of mutant P709A/ F1086A yielded the 150-kDa immature form of P-gp as the major product (Fig. 3F). Login to comment 161 ABCB1 p.Phe1086Ala It is possible that the F1086A mutation inhibited activity by disrupting coupling between the NBDs and TMDs. Login to comment 162 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling. Login to comment 163 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys The T333C and L975C mutations are located at the extracellular ends of TM segments 6 and 12, respectively (Fig. 3, A and B). Login to comment 164 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys Mutant T333C/L975C can be cross-linked when intact cells expressing the mutant are treated with BMOE cross-linker (30). Login to comment 165 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys Mutant T333C/L975C, however, does not show cross-linking if membranes containing the mutant are treated only with BMOE (Fig. 4A). Login to comment 170 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala 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 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B). Login to comment 172 ABCB1 p.Phe1086Ala These results suggest that F1086A disrupted NBD/TMD coupling. Login to comment 173 ABCB1 p.Phe1086Ala Replacement of Phe-1086 with Bulky Hydrophobic Amino Acids Yields Active Mutants-Mutant F1086A was inactive (Fig. 3C). Login to comment 175 ABCB1 p.Phe1086Asp ABCB1 p.Phe1086Arg Fig. 5A shows that the F1086D or F1086R mutations inhibited maturation of P-gp. Login to comment 176 ABCB1 p.Phe1086Trp ABCB1 p.Phe1086Leu ABCB1 p.Phe1086Tyr By contrast, those containing mutations of hydrophobic residues (F1086Y, F1086L, or F1086W) did not inhibit maturation. Login to comment 177 ABCB1 p.Phe1086Asp ABCB1 p.Phe1086Arg Mutants F1086D and F1086R could still be rescued if they were expressed in the presence of a drug substrate (Fig. 5B). Login to comment 179 ABCB1 p.Phe1086Asp ABCB1 p.Phe1086Arg Mutants F1086D and F1086R were first expressed in the presence of cyclosporine A to promote maturation before isolating the histidine-tagged protein. Login to comment 182 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala F1086A inhibits ATP-dependent cross-linking at the extracellular surface. Login to comment 187 ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala Mutant A266D/ F1086A showed little activity (Fig. 6C). Login to comment 195 ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs. Login to comment 212 ABCB1 p.Phe1086Asp ABCB1 p.Phe1086Arg ABCB1 p.Phe1086* A, F1086X mutants were expressed in the absence of drug substrates. B, processing mutants F1086D and F1086R were expressed in the presence (af9;) or absence (afa;) of cyclosporine A (Cyclo), and whole cell SDS extracts were subjected to immunoblot analysis. Login to comment 213 ABCB1 p.Phe1086* The positions of mature (170 kDa) and immature (150 kDa) P-gps are indicated. C, ATPase activities of histidine-tagged wild-type P-gp and F1086X mutants were measured in the presence of verapamil. Login to comment 214 ABCB1 p.Phe1086Asp ABCB1 p.Phe1086Arg Histidine-tagged mutants F1086D and F1086R were first expressed in the presence of cyclosporine A to promote maturation of P-gp before isolation of the protein by nickel-chelate chromatography. Each value is the mean afe; S.D. (n afd; 3). Login to comment 216 ABCB1 p.Phe1086Ala Replacement of Ala-266 with aromatic residues restores F1086A activity. Login to comment 217 ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala 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 ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys ABCB1 p.Thr333Cys ABCB1 p.Thr333Cys ABCB1 p.Phe1086Ala ABCB1 p.Phe1086Ala 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 ABCB1 p.Phe1086Ala In addition, activity of the F1086A mutant could be restored if Ala-266 was replaced with an aromatic amino acid. Login to comment