ABCMdb

PMID: 17627029

Zolnerciks JK, Wooding C, Linton KJ
Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein.
FASEB J. 2007 Dec;21(14):3937-48. Epub 2007 Jul 12., [PubMed]

Sentences

No. Mutations Sentence Comment

54 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys Novel cysteine codons were introduced into pMDR-cys- by site-directed mutagenesis (QuikChange multisite; Stratagene, La Jolla, CA, USA) using the oligonucleotides L443C, 5Ј-GTCCAGCTGATGCAGCGCTGCTATGACCCCACAG-3Ј; S474C, 5Ј-CTACGGGAAATCATTGGCGTCGTGTGTCAGGA- ACCTGTATTG-3Ј; R905C, 5Ј-GCAATAGAAAACTTCTGTAC- Figure 1. Login to comment 86 ABCB1 p.Ser909Cys AGTTGTTTCTTTGAC-3Ј; and S909C, 5Ј-CCGAACCGTTG- TTTGTCTCACCCAGGAGCAGAAGTTTG-3Ј. Login to comment 87 ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln The E556Q mutant was generated by mutation of the wild-type P-glycoprotein using the oligonucleotide E556Q, 5Ј- C C C C A A G A T C C T C C T G C T T G A T C A G G C C A C G - TCAGCCTTGG-3Ј. Login to comment 139 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys The residues in ICL4 and NBD1 predicted to be in close proximity were replaced individually and also in the following pairwise combinations; Leu443Cys with Ser909Cys (L443CϩS909C), and Ser474Cys with Arg905Cys (S474CϩR905C). Login to comment 151 ABCB1 p.Glu556Gln Untransfected and mock-transfected cells and cells expressing an inactive form of P-glycoprotein [the E556Q mutant (36)] are indistinguishable from each other and accumulate drug at a high rate, demonstrating that the efflux observed in the cells transfected with pMDR-wt is due to P-gp activity. Login to comment 155 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys Each of the single cysteine substitution mutants L443C, S474C, R905C, and S909C retain significant levels of drug transport activity, as do the L443CϩS909C and S474CϩR905C double mutants (Fig. 3B, C). Login to comment 169 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys B) Histogram of cell surface expression of P-glycoproteins L443C, S474C, R905C, S909C and double mutants L443CϩS909C and S474CϩR905C are shown as indicated. Login to comment 173 ABCB1 p.Ser909Cys However, to rule out the possibility that the gel retardation is the product of intermolecular cross linking between, for example, L443C on one P-glycoprotein with S909C on a second molecule, we cotransfected HEK293T cells with plasmids encoding both the single cysteine forms of the double mutants. Login to comment 180 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys Taken together with the results of the cross-linking, the simplest interpretation of these data is that L443C is in close proximity to S909C and that S474C is in close proximity to R905C in the tertiary structure of monomeric P-glycoprotein, entirely consistent with the hypothesis based on the crystal structure of Sav1866. Login to comment 183 ABCB1 p.Ser909Cys Both treatments caused a reproducible reduction in the level of cross-linking between L443C and S909C (fewer slow migrating P-glycoprotein species and more noncrosslinked species) with each of the three chemical cross-linkers, although the effect seems more pronounced with BMH (Fig. 7A). Login to comment 184 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys Repetition with the mutant P-glycoprotein with a cysteine pair at positions S474C and R905C showed no evidence of conformational change (Fig. 7B), providing a convenient negative control for non-specific effects of the added nucleotides. Login to comment 189 ABCB1 p.Glu556Gln In (A) the initial rates of Bodipy-taxol uptake by cells expressing E556Q and mock-transfected cells (dashed, blue and green lines, respectively) are described by the right-hand ordinate. Login to comment 207 ABCB1 p.Ser474Cys ABCB1 p.Glu556Gln ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys Efflux activity of cysteine mutants wt Pgp-cys- L443C S909C L443CϩS909C S474C R905C S474CϩR905C E556Q Mock Mean % activity 100 100.8 100.7 99.9 100.6 99.5 96.9 101.2 8.6 0.0 (Ϯse) Ϯ 0.6 Ϯ 0.8 Ϯ 0.4 Ϯ 0.5 Ϯ 1.4 Ϯ1.7 Ϯ0.9 Ϯ1.2 Ϯ 11.9 Ϯ 8.9 n 6 6 2 2 4 2 4 2 6 4 The efflux activity of the mutants generated in this study is presented as a percentage of the drug efflux activity of wild-type P-glycoprotein (wt), as described in Materials and Methods. Login to comment 209 ABCB1 p.Glu566Gln Similarly, the activity of the E566Q mutant was indistinguishable from the efflux activity of cells in the absence of P-glycoprotein (mock transfected cells). Login to comment 212 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys SDS-PAGE and Western analyses of P-glycoproteins with novel cysteines introduced into NBD1 (L443C and S474C) and/or ICL4 of TMD2 (R905C and S909C), as indicated. Login to comment 219 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys L443C P-glycoprotein was coexpressed in cells with S909C P-glycoprotein, and likewise S474C P-glycoprotein was coexpressed with R905C P-glycoprotein. Login to comment 220 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys SDS-PAGE and Western analyses showed no evidence of intermolecular cross-linking (lack of a slow mobility P-glycoprotein species) between the L443C on one protein and S909C on another, or between S474C and R905C on different P-glycoproteins. Login to comment 245 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys ABCB1 p.Ser909Cys SDS-PAGE and Western analyses of P-glycoprotein double mutants with novel cysteines introduced at (A) L443C and S909C, or (B) S474C and R905C. Login to comment 257 ABCB1 p.Ser909Cys Evidence in support of the interaction between ICL4 and NBD1 being a site of energy transduction through which the ATP and drug binding sites are coupled allosterically is provided by the modulation of the cross-linking efficiency between L443C and S909C by the nonhydrolysable ATP analog AMP-PNP, and also by ADP in the presence of vanadate (ADP.Vi) (Fig. 7A). Login to comment 264 ABCB1 p.Ser909Cys While it is unlikely that all the P-glycoprotein molecules in a membrane fraction would be accessible to nucleotide (the NBDs of some in the fraction are likely to be intravesicular), the data suggest that in the presence of AMP-PNP or ADP.Vi the L443C and S909C cysteines are less available for cross-linking. Login to comment 266 ABCB1 p.Ser474Cys ABCB1 p.Arg905Cys The differential response of the two cysteine pairs (cross-linking in the S474C and R905C double mutant appears insensitive to added nucleotide), suggests that S474 and R905 may move in tandem during the ATP catalytic cycle, if they move at all. Login to comment