ABCMdb

PMID: 26507655

Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PubMed]

Sentences

No. Mutations Sentence Comment

63 ABCB4 p.Phe770Cys Disulfide Cross-linking Analysis-Cys-less P-gp mutants containing pairs of cross-linkable cysteines in the TM segments (A80C(TM1)/R741C(TM7), I299C(TM5)/F770C (TM8), and T333C(TM6)/L975C(TM12)), intracellular loops (ICLs) (L175C(ICL1)/N820C(ICL3), A259C(ICL2)/ W803C(ICL3), and A266C(ICL2)/F1086C(NBD2)), or the NBDs (C431(NBD1)/L1176C(NBD2), L531C(NBD1)/ C1074(NBD2), and P517C(NBD1)/I1050C(NBD2)) were transiently expressed in HEK 293 cells at reduced temperature (30 &#b0;C) to promote maturation. Login to comment 65 ABCB1 p.Phe1086Cys ABCB1 p.Trp803Cys ABCB1 p.Arg741Cys ABCB4 p.Phe770Cys ABCB4 p.Ile299Cys Membranes were prepared and samples were incubated at 0 &#b0;C (A80C/R741C, A259C/W803C, I299C/F770C, and A266C/ F1086C) or 20 &#b0;C (L175C/N820C, C431/L1176C, and L521C/C1074) for 10 min in the presence or absence of 0.5 Mapping the P-glycoprotein Tariquidar-binding Site 29390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 49ߦDECEMBER 4, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 11, mM copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 òe;M (for TM segment cysteine mutants) or 1 òe;M tariquidar (for the ICL and NBD cysteine mutants). Login to comment 66 ABCB1 p.Thr333Cys Mutant T333C/L975C was cross-linked in whole cells at 20 &#b0;C with 0.5 mM BMOE (bismaleimidoethane) cross-linker (Thermo Fisher Scientific, Burlington, ON) in the absence or presence of 0.25 òe;M tariquidar (30). Login to comment 67 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Mutant P517C/I1050C was cross-linked in membranes at 0 &#b0;C with 50 òe;M 1,4-butanediyl bismethanethiosulfonate (BMTS) in the absence or presence of 1 òe;M tariquidar (30). Login to comment 69 ABCB1 p.Arg741Cys The reaction mixtures were then subjected to SDS-PAGE (6.5% (w/v) polyacrylamide gels and 7% gels for mutant A80C/R741C) and immunoblot analysis with a rabbit polyclonal antibody against P-gp. Login to comment 112 ABCB1 p.Trp803Cys Accordingly, a Cys-less P-gp (35) was used to generate mutants that contained double cysteines at the interfaces involving TM segments of TMD1 and TMD2 (A80C(TM1)/ R741C(TM7) (29), I299C(TM5)/F770C(TM8) (36), and T333C(TM6/L975C(TM12) (30), or ICL2 and ICL3 (L175C(ICL1)/N820C(ICL3) (23), A259C(ICL2)/W803C- (ICL3) (37), and A266C(ICL2/F1086C(NBD2) (38) (Fig. 1A). Login to comment 118 ABCB1 p.Thr333Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The conditions for cross-linking mutants T333C/L975C and P517C/I1050C, however, were different. Login to comment 119 ABCB1 p.Thr333Cys Mutant T333C/L975C was cross-linked with BMOE using intact cells because ATP hydrolysis is required for cross-linking (41). Login to comment 120 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Cross-linking of mutant P517C/I1050C was performed on membranes using BMTS cross-linker (30). Login to comment 156 ABCB1 p.Met69Arg For example, we showed that an M69R mutation specifically blocked rescue of a P-gp G251V processing mutant with verapamil but not rescue with cyclosporine A, vinblastine, or rhodamine B (43). Login to comment 164 ABCB1 p.Tyr307Arg By contrast, the Y307R mutation inhibited rescue with both tariquidar and cyclosporine A. Login to comment 167 ABCB1 p.Tyr307Arg ABCB1 p.Ile868Arg A, whole cell SDS extracts of cells expressing P-gp processing mutant G251V, G251V/I868R, or G251V/Y307R in the presence of various concentrations of tariquidar or cyclosporine A were subjected to immunoblot analysis. Login to comment 175 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Ile306Arg ABCB1 p.Met68Arg ABCB1 p.Met69Arg ABCB1 p.Phe72Arg ABCB1 p.Tyr307Arg ABCB1 p.Phe303Arg In the N-terminal TMD1 domain, the largest number of arginine mutations predicted to line the drug-binding pocket that inhibited tariquidar rescue were located in TM1 (H61R, G64R, L65R, M68R, M69R, and F72R) and TM5 (F303R, I306R, Y307R, S309R, and Y310R) (Fig. 4, A and E). Login to comment 176 ABCB1 p.Phe343Arg ABCB1 p.Phe336Arg ABCB1 p.Ala129Arg One arginine mutation predicted to line the drug-binding pocket inhibited rescue in TM2 (A129R) (Fig. 4B) and two arginines predicted to line the drug-binding pocket in TM6 (F336R and F343R) were FIGURE 4. Login to comment 188 ABCB1 p.Thr945Arg ABCB1 p.Gly872Arg ABCB1 p.Val982Arg ABCB1 p.Phe942Arg ABCB1 p.Ile868Arg ABCB1 p.Phe978Arg ABCB1 p.Met949Arg ABCB1 p.Tyr950Arg ABCB4 p.Phe732Arg ABCB1 p.Gln946Arg ABCB1 p.Val865Arg ABCB4 p.Phe728Arg ABCB4 p.Gln725Arg ABCB1 p.Tyr953Arg These were in TM7 (Q725R, F728R, and F732R) (Fig. 6A), TM10 (V865R, I868R, and G872R) (Fig. 6D), TM11 (F942R, T945R, Q946R, M949R, Y950R, and Y953R) (Fig. 6E), and TM12 (L975R, F978R, and V982R) (Fig. 6F). Login to comment 193 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Ile306Arg ABCB1 p.Met68Arg ABCB1 p.Met69Arg ABCB1 p.Thr945Arg ABCB1 p.Gly872Arg ABCB1 p.Val982Arg ABCB1 p.Phe942Arg ABCB1 p.Ile868Arg ABCB1 p.Ile868Arg ABCB1 p.Phe303Arg ABCB1 p.Gln946Arg ABCB4 p.Phe728Arg ABCB4 p.Gln725Arg It was found that 16 of the 28 mutants resembled the G251V/I868R mutant as expression in the presence of 5 òe;M cyclosporine A yielded mature P-gp as the major product in TM1 (H61R, G64R, L65R, M68R, and M69R), TM5 (F303R, I306R, and S309R), TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 (F942R, T945R, and Q946R), and TM12 (V982R) (Fig. 7). Login to comment 194 ABCB1 p.Phe343Arg ABCB1 p.Phe72Arg ABCB1 p.Phe336Arg ABCB1 p.Tyr307Arg ABCB1 p.Phe978Arg ABCB1 p.Met949Arg ABCB1 p.Tyr950Arg ABCB4 p.Phe732Arg ABCB1 p.Val865Arg ABCB1 p.Tyr953Arg ABCB1 p.Ser952Arg The other 12 mutants in TM1 (F72R), TM5 (Y307R and Y310R), TM6 (F336R and F343R), TM7 (F732R), TM10 (V865R), TM11 (M949R, Y950R, S952R, and Y953R), and TM12 (L975R and F978R) were not rescued by cyclosporine A (Fig. 7). Login to comment 212 ABCB1 p.Ile306Arg ABCB1 p.Met68Arg ABCB1 p.Met69Arg ABCB1 p.Phe72Arg ABCB1 p.Thr945Arg ABCB1 p.Gly872Arg ABCB1 p.Val982Arg ABCB1 p.Phe942Arg ABCB1 p.Phe336Arg ABCB1 p.Tyr307Arg ABCB1 p.Ile868Arg ABCB1 p.Met949Arg ABCB4 p.Phe732Arg ABCB4 p.Phe728Arg ABCB1 p.Ser952Arg Seventeen of the 30 G251V/arginine mutants (M68R, M69R, and F72R in TM1; I306R, Y307R, S309R, and Y310R in TM5; F336R in TM6; F728R and F732R in TM7; I868R and G872R in TM10; F942R, T945R, M949R, and S952R in TM11; and V982R in TM12) that could not be rescued with tariquidar showed little or no stimulation of ATPase activity with tariquidar (Fig. 8A). Login to comment 213 ABCB1 p.Phe978Arg Mutants L65R(TM1), A129R(TM2), F732R(TM7), Y950R(TM11), Y953R(TM11), and F978R- (TM12) showed partial activity (about 25-50% of wild-type activity), whereas the maximal tariquidar-stimulated ATPase activity of mutants H61R(TM1), G64R(TM1), F303R(TM5), F343R(TM6), Q725R(TM7), V865R(TM10, Q946R(TM11), and L975R(TM12) was about 50-100% of wild-type enzyme. Login to comment 283 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Ile306Arg ABCB1 p.Phe343Arg ABCB1 p.Met68Arg ABCB1 p.Thr945Arg ABCB1 p.Phe942Arg ABCB1 p.Ala129Arg ABCB1 p.Tyr950Arg ABCB1 p.Gln946Arg We identified 13 additional arginine mutations (H61R, G64R, L65R, and M68R in TM1; A129R in TM2; I306R and S309R in TM5; F343R in TM6; F942R, T945R, Q946R, and Y950R in TM11; and L975R in TM12) (Fig. 11A) that were not predicted to lie within 4.5-&#c5; of the predicted site 3 tariquidar-binding site (9). Login to comment 313 ABCB1 p.Gly64Arg ABCB1 p.Leu65Arg ABCB1 p.Thr945Arg ABCB1 p.Phe942Arg ABCB1 p.Tyr950Arg ABCB1 p.Gln946Arg Therefore, arginines predicted to lie outside of the tariquidar-binding site in TM1 and TM11 in the docking studies (H61R, G64R, L65R, and M68 in TM1; F942R, T945R, Q946R, and Y950R in TM11) might alter interactions between TM1 and TM11. Login to comment