ABCMdb

PMID: 24523403

Loo TW, Clarke DM
Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.
J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 &#c5; (ॷ-carbons). Login to comment 12 ABCB1 p.Leu175Cys The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 &#c5;, respectively. Login to comment 47 ABCB1 p.Leu175Cys To address these questions, we used cross-linkers of various lengths to cross-link a P-gp mutant that contained reporter cysteines at positions L175C (N-half) and N820 (C-half) in a Cys-less background. Login to comment 48 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys The L175C and N820C reporter cysteines were selected because they are located at positions predicted to undergo large changes in distance between the closed (12 &#c5; between ॷ-carbons) and open (about 30 &#c5; between ॷ-carbons) conformations. Login to comment 49 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys In addition, modifications to L175C and N820C are not expected to have a major impact on activity because they reside outside the drug- and ATP-binding sites. Login to comment 50 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys We previously showed that L175C/N820C could be efficiently cross-linked with the short cross-linker 1,4-butanediyl bismethanethiosulfonate (M4M) that can span a distance of 7.5 &#c5; (about 13 &#c5; between ॷ-carbons) to activate ATPase activity (40). Login to comment 51 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Here, we tested the effects of cross-linking mutant L175C/ N820C at 10 different distances between the halves. Login to comment 52 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys It was found that linkage of L175C to N820C with cross-linkers predicted to span a distance of less than 20 &#c5; highly activated P-gp ATPase activity (over 10-fold), but cross-linking with longer cross-linkers yielded P-gps with low ATPase activity. Login to comment 55 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Mutations L175C and N820C were introduced into Cys-less P-gp as described previously (42). Login to comment 60 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Disulfide Cross-linking Analysis-The double cysteine mutant L175C/N820C was transiently expressed in HEK 293 cells. Login to comment 66 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Intramolecular disulfide cross-linking between L175C and N820C can be detected because the cross-linked product migrates with a slower mobility on SDS-polyacrylamide gels (47). Login to comment 68 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of L175C/N820C with M1M or oxidant (copper phenanthroline) was performed using P-gp isolated by nickel-chelate chromatography. Login to comment 81 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys P-gp ATPase Activity Is Highly Activated When Residues L175C and N820C Are Cross-linked in Close Proximity-In a previous study, we found that cross-linking the two homologous halves through formation of a direct disulfide bond between A259C in intracellular helix 2 (IH2) and W803C in IH3 inhibited activity (49). Login to comment 82 ABCB1 p.Trp803Cys ABCB1 p.Ala259Cys It is possible that the A259C/W803C cross-link inhibited activity because these residues are located within IH segments that are predicted to form critical TMD/ NBD contact points. Login to comment 83 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys Because cysteines at positions L175C and N820C are located away from the IH TMD/NBD contact points (Fig. 1), cross-linking L175C and N820C in close proximity might not disrupt TMD/NBD interactions. Login to comment 84 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys The L175C or N820C mutations did not appear to affect activity of P-gp because the drug-stimulated ATPase activity of the L175C/N820C mutant was very similar to the Cys-less parent (42). Login to comment 86 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of mutant L175C/N820C was low (b0d;50% cross-linking) when membranes prepared from transfected cells were treated with oxidant (copper phenanthroline) even at 37 &#b0;C (data not shown). Login to comment 88 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Accordingly, histidine-tagged mutant L175C/N820C was expressed in HEK 293 cells, isolated by nickel chelate chromatography and treated with oxidant. Login to comment 89 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys In the presence of dodecyl maltoside detergent, mutant L175C/ N820C could be efficiently cross-linked with copper phenanthroline (Fig. 2, A and B). Login to comment 101 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of L175C and N820C in close proximity highly activates P-gp basal ATPase activity. Login to comment 102 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys A, purified histidine-tagged mutant L175C/N820C was treated with (af9;) or without (afa;) copper phenanthroline (CuP) or M1M cross-linker. Login to comment 105 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys B, amount of cross-linked protein relative to total P-gp was quantitated from three different transfections afe; S.D. C, ATPase activities of mutant L175C/N820C were determined in the absence (afa;) or presence (af9;) of verapamil (Ver) before (None) and after cross-linking with copper phenanthroline or M1M. Login to comment 107 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys To test for the effects of cross-linking on activity, isolated L175C/N820C was treated with or without 0.5 mM copper phenanthroline or 0.009 mM M1M. Login to comment 112 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Activation of mutant L175C/N820C basal ATPase activity with M1M was higher than previously observed using membranes (42) suggesting that the efficiency of cross-linking was improved using purified P-gp. Login to comment 115 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys P-gp ATPase Activity Is Not Highly Activated When Residues L175C and N820C Are Cross-linked with Long Cross-linkers- The next step was to test the effects of cross-linking with MTS cross-linkers that could span longer distances such as M2M (5.2 &#c5;), M4M (7.8 &#c5;), M5M (8.8 &#c5;), M6M (10.4 &#c5;), M8M (13 &#c5;), M11M (16.9 &#c5;), M14M (19 &#c5;) and M17M (22 &#c5;) (45). Login to comment 117 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys For example, we used 0.009 mM M1M to treat L175C/N820C because we previously showed that cross-linking efficiency was sharply reduced at lower concentrations (0.003 mM) or higher concentrations (0.027 mM) (42). Login to comment 118 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Accordingly, membranes prepared from cells expressing mutant L175C/N820C were treated with various concentrations of MTS cross-linkers with various lengths of spacer arms for 10 min at 20 &#b0;C, and samples were subjected to immunoblot analysis. Login to comment 127 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Membranes prepared from cells expressing mutant L175C/ N820C were first treated for 10 min at 20 &#b0;C in the absence or presence of 0.027 mM MTS cross-linkers of different lengths. Login to comment 129 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Because mutant L175C/N820C was efficiently cross-linked with the MTS cross-linkers, we tested whether cross-linking affected activity. Login to comment 131 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of L175C/N820C P-gp with various concentrations of M2M and M17M cross-linker. Login to comment 132 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Membranes prepared from HEK 293 cells expressing mutant L175C/N820C were treated with various concentrations (0-2.2 mM) of M2M (A and B) or M17M (C and D) cross-linker. Login to comment 136 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Activation Switch for P-gp ATPase Activity MARCH 21, 2014ߦVOLUME 289ߦNUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8487 membranes and assayed for ATPase activity in the presence or absence of saturating concentrations (0.4 mM) of verapamil. Fig. 5 shows that untreated mutant L175C/N820C isolated from membranes exhibited about a 12-fold increase in verapamil-stimulated ATPase activity. Login to comment 139 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys By contrast, treatment of mutant L175C/ N820C with the intermediate size MTS cross-linkers (M2M, M4M, M5M, M6M, and M8M) that span maximum distances of about 5.2-13 &#c5; (distance of about 10.6-18.6 &#c5; between the ॷ-carbons) highly activated ATPase activity in the absence of verapamil. Login to comment 142 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys It appeared that cross-linking of mutant L175C/N820C with the intermediate size cross-linkers locked P-gp in an activated state resulting in high levels of ATPase activity in the absence or presence of drug substrates. Login to comment 143 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys A quite different effect was observed when mutant L175C/ N820C was treated with the long cross-linkers M11M, M14M, and M17M that could span maximum distances of about 16.9, 19, and 22 &#c5; in length, respectively (span distances of about 22 to 28 &#c5; between the ॷ-carbons) (Fig. 5). Login to comment 145 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of mutant L175C/N820C with the intermediate size MTS cross-linkers like M2M increased the basal ATPase activity of the mutant (Fig. 5). Login to comment 147 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys To test these possibilities, a series of control experiments were carried out with L175C/N820C using M2M. Login to comment 150 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys We compared the effects of treating mutant L175C/N820C with low (0.027 mM; promotes cross-linking) or high (3 mM; likely labels each cysteine with a separate M2M molecule) concentrations of M2M on ATPase activity. Login to comment 154 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Next, mutant histidine-tagged P-gps were constructed that contained only the L175C or N820C mutation alone to test if modification of only one cysteine had an impact on ATPase activity. Login to comment 159 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of mutant L175C/N820C with cross-linkers of various lengths. Login to comment 160 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys A, membranes prepared from HEK 293 cells expressing mutant L175C/N820C were treated without (None) or with MTS cross-linkers of various lengths (MXM). Login to comment 164 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ATPase activity of mutant L175C/N820C cross-linked with various MXM cross-linkers. Login to comment 165 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Membranes were prepared from HEK 293 cells expressing mutant histidine-tagged L175C/N820C and cross-linked with various MXM cross-linkers (X afd; 2-17). Login to comment 169 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys These results show that activation of mutant L175C/N820C basal ATPase activity by M2M was due to cross-linking between the two cysteines. Login to comment 173 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys In agreement with the EPR and simulation studies (55), we found that the presence of ATP did not promote the formation of a more closed structure as it did not promote L175C/N820C cross-linking (42). Login to comment 174 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys We also found that verapamil did not promote or inhibit cross-linking of L175C/N820C (42). Login to comment 179 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys The experiments presented here show that human L175C/ N820C P-gp remains active when it is trapped in conformations with a wide range of separations between the halves (about 6-30 &#c5;). Login to comment 192 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys Activity of L175C/N820C, Cys-less, L175C, and N820C P-gp treated with various concentrations of M2M. Login to comment 193 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys A, histidine-tagged mutant L175C/N820C was isolated from membranes treated was treated without (None), low (0.027 mM), or high (3 mM) concentrations of M2M and ATPase activity determined in the absence (afa;) or presence (af9;) of verapamil (Ver). Login to comment 194 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys B, membranes expressing histidine-tagged mutant L175C/N820C, Cys-less, L175C, or N820C P-gp were treated without (afa;) or with (af9;) 0.027 mM M2M. Login to comment 197 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys Correlation of ATPase activity of L175C/N820C treated with cross-linkers of various lengths and the distance between the ॷ-carbons of L175C and N820C. Login to comment 198 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys The activity of mutant L175C/N820C cross-linked with MXM cross-linker (M1M (2), M2M (3), M4M (4), M5M (5), M6M (6), M8M (7), M11M (8), M14M (9), or M17M (10)) was compared with that treated with copper phenanthroline (1). Login to comment 207 ABCB1 p.Ala295Cys ABCB1 p.Trp803Cys The P-gp molecular dynamics simulation study (64) may also provide an explanation for the inhibition of ATPase activity previously observed when cysteines A295C (in IH2 that connects TM segments 4 and 5) and W803C (in IH3 that connects TM segments 9 and 10) were directly cross-linked (49). Login to comment 209 ABCB1 p.Ala295Cys ABCB1 p.Trp803Cys It is possible that direct cross-linking of A295C (in IH2 that connects TM segments 4 and 5) to W803C in IH3 would interfere with straightening of TM segments 4 and 5 to trap P-gp in an inactive conformation. Login to comment 211 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Could P-gp still transport drug substrates when L175C/ N820C is cross-linked with short (M2M to M8M) cross-linkers? Login to comment 212 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Unfortunately, it was not possible to use whole cell transport or drug resistance assays because cross-linking could not be detected when cells expressing the L175C/N820C mutant were treated with the MTS cross-linkers.3 The lack of cross-linking in whole cells could be due to the presence of intracellular thiol reductants such as glutathione that would not only react with the MTS cross-linkers but also reduce any disulfide bond. Login to comment 214 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys We predict, however, that P-gp cross-linked at L175C/ N820C with short cross-linkers should be able to bind substrates with relatively high affinity because it was reported that P-gp trapped in the closed vanadate-trapped transition state had similar affinity for the drug substrates verapamil, Hoechst 33342, cyclosporin A, vinblastine, colchicine, rhodamine B, and doxorubicin as in the relaxed (open) state (66). Login to comment