ABCMdb

PMID: 22700974

Loo TW, Bartlett MC, Detty MR, Clarke DM
The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. Login to comment 13 ABCB1 p.Leu975Cys ABCB1 p.Thr333Cys Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. Login to comment 91 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively. Login to comment 93 ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys To address whether trapping the NBDs in close proximity (closed conformation) would activate or inhibit ATPase activity, cysteines were introduced at locations in the central part of NBD1 (P517C) and the N-terminal region of NBD2 (I1050C) of Cys-less P-gp predicted to be outside the catalytic sites but close to the LSGGQ (residues 531-535) and Walker A sites (residues 1070-1077) of NBD1 and NBD2, respectively. Login to comment 94 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The P517C/I1050C mutant was expressed in HEK 293 cells. Login to comment 95 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys In the open conformation P517C and I1050C are predicted to be far apart (22.9 Å; measured from the ␣ carbons) but close (7.8 Å) in the closed conformation. Login to comment 96 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The P517C/I1050C mutant was expressed in HEK 293 cells. Login to comment 97 ABCB1 p.Pro517Cys To test the effect of covalently linking NBD1 and NBD2 on catalytic activity, ATPase activity of mutant P517C/NI1050C was measured in the presence or absence of verapamil before and after cross-linking with the short M4M cross-linker. Login to comment 99 ABCB1 p.Pro517Cys To test the effect of covalently linking NBD1 and NBD2 on catalytic activity, ATPase activity of mutant P517C/NI1050C was measured in the presence or absence of verapamil before and after cross-linking with the short M4M cross-linker. Login to comment 104 ABCB1 p.Ala935Cys ABCB1 p.Thr333Cys ABCB1 p.Ser909Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys ABCB1 p.Leu443Cys ABCB4 p.Leu975Cys The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment 105 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Linkage of the NBDs Activates P-gp ATPase Activity 26808 activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 af9; 0.2 òe;mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 af9; 0.3 òe;mol Pi/min/mg P-gp). Login to comment 106 ABCB1 p.Ala935Cys ABCB1 p.Thr333Cys ABCB1 p.Ser909Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys ABCB1 p.Leu443Cys ABCB4 p.Leu975Cys The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment 107 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Linkage of the NBDs Activates P-gp ATPase Activity 26808 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 32•AUGUST 3, activity mutant P517C/I1050C was highly activated (11.5-fold) by verapamil and exhibited about 80% (1.5 ϩ 0.2 ␮mol Pi/min/mg P-gp) of the verapamil-stimulated ATPase activity of Cys-less P-gp (1.9 ϩ 0.3 ␮mol Pi/min/mg P-gp). Login to comment 108 ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys Cross-linking of P517C/I1050C with M4M increased its basal ATPase activity (14-fold higher than the basal ATPase activity of untreated P-gp) (Fig. 2B). Login to comment 109 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B). Login to comment 110 ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys The enhanced activity of M4M cross-linked P517C/I1050C was not due to carry over of M4M during purification because M4M had little effect on Cys-less P-gp ATPase activity (Fig. 2B). Login to comment 111 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys Reduction of M4M cross-linked P517C/I1050C with dithiothreitol decreased its activity back to basal levels (Fig. 2B). Login to comment 112 ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys The large increase in basal ATPase activity observed when mutant P517C/I1050C was cross-linked with the short M4M cross-linker appeared to be caused by trapping of the NBDs in close proximity rather than nonspecific cross-linking effects because treatment with the long M17M cross-linker caused a much smaller increase (about 2-fold) in basal ATPase activity (Fig. 2B). Login to comment 113 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Cross-linking of ICL1 (TMD1) and ICL3 (TMD2) in Close Proximity Also Activates P-gp ATPase Activity-To test if cross-linking of other segments predicted to undergo large conformational changes during the reaction cycle would also activate P-gp ATPase activity, we performed cross-linking studies on mutant D177C/N820C. Login to comment 114 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The cross-linking results observed with mutant P517C/ I1050C suggest that covalently linking NBD1 and NBD2 in close proximity with M4M mimics activation of ATPase activity with compounds that highly activate P-gp ATPase activity. Login to comment 115 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Cross-linking of ICL1 (TMD1) and ICL3 (TMD2) in Close Proximity Also Activates P-gp ATPase Activity-To test if cross-linking of other segments predicted to undergo large conformational changes during the reaction cycle would also activate P-gp ATPase activity, we performed cross-linking studies on mutant D177C/N820C. Login to comment 117 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M. Login to comment 120 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys Membranes prepared from cells expressing mutant D177C/ N820C were treated with M4M and M17M. Login to comment 122 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment 123 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys A, membranes prepared from HEK 293 cells expressing the NBD1/ NBD2 mutant P517C/I1050C were treated without (Control) or with cross-linkers M4M or M17M. Login to comment 125 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment 126 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). Login to comment 129 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). Login to comment 132 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys C, membranes expressing mutants D177C/N820C or L175C/N820C were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment 133 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B). Login to comment 134 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Prior to cross-linking, mutant D177C/N820C showed verapamil-stimulated ATPase activity that was similar to the Cys-less parent. Login to comment 135 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys The basal ATPase activity of the M4M cross-linked D177C/N820C mutant was similar to that of uncross-linked mutant assayed in the presence of verapamil (Fig. 3B). Login to comment 136 ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys It was found that the effect of cross-linking on ATPase of mutant D177C/N820C (Fig. 3C) was very similar to those observed for mutant P517C/I1050C (Fig. 2B). Login to comment 137 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys Prior to cross-linking, mutant D177C/N820C showed verapamil-stimulated ATPase activity that was similar to the Cys-less parent. Login to comment 138 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys The basal ATPase activity of the M4M cross-linked D177C/N820C mutant was similar to that of uncross-linked mutant assayed in the presence of verapamil (Fig. 3B). Login to comment 139 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys No further stimulation of ATPase activity was observed when the M4M cross-linked D177C/N820C was assayed in the presence of verapamil. Login to comment 140 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 &#b0;C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment 141 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment 142 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys One possibility is that a separate M1M molecule preferentially modifies each cysteine of mutant D177C/ N820C as it was found that cross-linking was reduced when the concentration of M1M was increased (data not shown). Login to comment 143 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 °C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment 144 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys ABCB1 p.Asp177Cys Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment 145 ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys One possibility is that a separate M1M molecule preferentially modifies each cysteine of mutant D177C/ N820C as it was found that cross-linking was reduced when the concentration of M1M was increased (data not shown). Login to comment 146 ABCB1 p.Leu175Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys We previously observed that mutant L175C/N820 only showed efficient cross-linking with a narrow concentration range of M1M (about 10-20 ␮M). Login to comment 147 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of L175C/N820C with M4M highly activated the basal ATPase activity of the mutant while cross-linking with M17M caused little activation. Login to comment 148 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil. Login to comment 150 ABCB1 p.Ala935Cys ABCB1 p.Ala935Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Asp177Cys We tested the effects of M4M and M17M cross-linking on the ATPase activity of mutant L175C/N820C and found that the effects were similar to those observed with mutant D177C/ N820C (Fig. 3C). Login to comment 151 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys Cross-linking of L175C/N820C with M4M highly activated the basal ATPase activity of the mutant while cross-linking with M17M caused little activation. Login to comment 152 ABCB1 p.Ser909Cys ABCB1 p.Ser909Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Leu443Cys The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil. Login to comment 154 ABCB1 p.Ala935Cys ABCB1 p.Ala935Cys ABCB1 p.Ala935Cys ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys ABCB1 p.Leu443Cys ATPase Activity Is Not Activated when the Homologous Halves Are Cross-linked at Locations Predicted Not to Undergo Large Distance Changes during the Open to Closed Conformational Change-Mutant C137/A935C contains natural Cys-137 in TM segment 2 (TMD1) and the A935C mutation in TM segment 11 (TMD2). Login to comment 156 ABCB1 p.Ser909Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Mutant L443C/S909C contains a cysteine (L443) in NBD1 and a cysteine (S909C) in the ICL4 loop that connects the cytoplasmic extensions of TM segments 10 and 11. Login to comment 158 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys ABCB1 p.Leu443Cys In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp. Login to comment 159 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Membranes prepared from cells expressing mutants C137/ A935C or L443C/S909C were treated with M4M cross-linker. Login to comment 161 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (af9;) or without (afa;) the M4M cross-linker. Login to comment 163 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys ABCB1 p.Leu443Cys These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation. Login to comment 166 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (ϩ) or without (-) the M4M cross-linker. Login to comment 168 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutants L443C/ S909C or C137/A935C were treated with or without (None) M4M cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment 189 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking. Login to comment 194 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys Mutants that showed activation of ATPase after cross-linking with M4M (P517C/I1050C and D177C/N820C) were tested for inhibition of cross-linking. Login to comment 195 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment 196 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment 197 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment 200 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (b0e; 90% efficiency) when intact cells were treated with BMOE. Login to comment 201 ABCB1 p.Thr333Cys ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys ABCB4 p.Leu975Cys Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment 202 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment 203 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment 204 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment 206 ABCB1 p.Thr333Cys ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys ABCB4 p.Leu975Cys Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (Ͼ 90% efficiency) when intact cells were treated with BMOE. Login to comment 207 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys To test for the effect of cross-linking on activity, histidine-tagged T333C/L975C P-gp was isolated by nickel-chelate chromatography before and after cross-linking with BMOE. Login to comment 210 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment 212 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys The results suggest that the stimulators may promote the closed conformation where the T333C and L975C residues are far apart. Login to comment 218 ABCB1 p.Thr333Cys ABCB4 p.Leu975Cys C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (afa;) or presence (af9;) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment 219 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (afa;) or presence (af9;) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis. Login to comment 225 ABCB1 p.Thr333Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB4 p.Leu975Cys C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment 226 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys D, membranes prepared from cells expressing mutant P517C/I1050C were pretreated without (None) or with 0.03 mM tariquidar (Tar) followed by incubation in the absence (-) or presence (ϩ) of 0.05 mM M4M cross-linker. Samples were then subjected to immunoblot analysis. Login to comment 229 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment 232 ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys An explanation for the increased activity is that P517C and I1050C are located at the N-terminal end and central segments of the NBDs close to the LSGGQ and Walker A sites of NBD1 and NBD2, respectively. Login to comment 233 ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys Cross-linking P517C/I1050C (or D177C/ N820C) with M4M would hold the Walker A and LSGGQ sites in close proximity (Fig. 7A) to increase the probability of generating the ATP-bound sandwich conformation and therefore increase the basal ATPase activity. Login to comment 236 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment 237 ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB4 p.Leu975Cys Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment 244 ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB4 p.Leu975Cys Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51). Login to comment 247 ABCB1 p.Leu65Cys ABCB1 p.Ile306Cys ABCB4 p.Phe728Cys For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold. Login to comment 248 ABCB1 p.Thr199Cys Covalent labeling of T199C (TM3) with a thiol-reactive derivative of rhodamine increased basal activity 7-fold (58). Login to comment 254 ABCB1 p.Leu65Cys ABCB1 p.Ile306Cys ABCB4 p.Phe728Cys For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold. Login to comment 255 ABCB1 p.Thr199Cys Covalent labeling of T199C (TM3) with a thiol-reactive derivative of rhodamine increased basal activity 7-fold (58). Login to comment 256 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln In support of this prediction, we observed that E556Q and E1201Q mutations to the catalytic carboxylates had different effects on cross-linking between TM segments 6 and 12 (59). Login to comment 257 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment 263 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln In support of this prediction, we observed that E556Q and E1201Q mutations to the catalytic carboxylates had different effects on cross-linking between TM segments 6 and 12 (59). Login to comment 264 ABCB1 p.Leu175Cys ABCB1 p.Asn820Cys ABCB1 p.Asn820Cys ABCB1 p.Ile1050Cys ABCB1 p.Pro517Cys ABCB1 p.Asp177Cys We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment 275 ABCB1 p.Ala935Cys The requirement for rotation or motion of the TM segments during the catalytic cycle may explain why direct cross-linking of the cysteines in mutant C137/A935C (TM2/TM11) (39) inhibited activity whereas cross-linking with M4M did not (this study). Login to comment 280 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle. Login to comment 282 ABCB1 p.Ala935Cys The requirement for rotation or motion of the TM segments during the catalytic cycle may explain why direct cross-linking of the cysteines in mutant C137/A935C (TM2/TM11) (39) inhibited activity whereas cross-linking with M4M did not (this study). Login to comment 287 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle. Login to comment 294 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 &#c5;, the average distance between ॷ carbons in a disulfide bond (64). Login to comment 297 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal. Login to comment 301 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 Å, the average distance between ␣ carbons in a disulfide bond (64). Login to comment 304 ABCB1 p.Ala935Cys ABCB1 p.Ser909Cys ABCB1 p.Leu443Cys Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal. Login to comment