ABCMdb

ABCB1 p.Gly346Cys

Predicted by SNAP2:	A: D (75%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (91%), N: D (85%), P: D (95%), Q: D (91%), R: D (91%), S: D (80%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Identification of residues in the drug-binding dom... J Biol Chem. 1999 Dec 10;274(50):35388-92. Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PMID:10585407]

Abstract [show]
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.

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194 The helices are also oriented to take into account the cross-linkable nature of residues 346 (TM6) and 989 (TM12) in the mutant G346C/G989C (25). Login to comment

[hide] Residue G346 in transmembrane segment six is invol... Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14. Storm J, O'Mara ML, Crowley EH, Peall J, Tieleman DP, Kerr ID, Callaghan R
Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein.
Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14., 2007-09-04 [PMID:17696319]

Abstract [show]
Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.

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66 Table 1: Mutagenic Oligonucleotide Primers Used to Generate TM6 Mutationsa mutation primer sequence 5'-3' diagnostic restriction digest S344C TTAATTGGGGCcTTTtGTGTTGGACAG + Eco 0109 I V345C TTAATTGGGGCaTTcAGTtgTGGACAGGCAT + Bsm I G346C F:GGGGCTTTTAGTGTTtGcCAGGCgTCTCCAAGCATTG +Bsa H I R:CAATGCTTGGAGAcGCCTGgCaAACACTAAAAGCCCC Q347C GCTTTTAGTGTTGGAtgcGCATCTCCAAG + Fsp I A348C GTTGGACAGtgcagcCCAAGCATTG + Bsg I S349C GGACAGGCATgcCCAAGTATTGAAGCA + Sph I A354C CAAGCATTGAAtgcTTTGCAAATG + Bsm I G360C CAAATGCAAGAtGcGCAGCTTATG + Fsp I a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates, or removes, the indicated restriction site. Login to comment
77 Mutants (G346C, Q347C, A348C, S349C, A354C, and G360C) in pBlueBac_4.5 (2 µg) were cotransfected into Spodoptera frugiperda (Sf9) cells with Bac-N-Blue DNA (0.25 µg) and Cellfectin (10 µg) in medium without FCS and antibiotics. Login to comment
117 The native P-gp model underwent an in silico G346C mutation and was further energy minimized to refine Transport actiVity ) cells in lower right quandrant cells in the lower and upper right quadrants × 100% the structure. Login to comment
118 The quality of the native P-gp and G346C mutant P-gp models was assessed using PROCHECK (41) and WHATIF (42). Login to comment
133 In addition, the data obtained for G346C displayed a shift from the lower right quadrant toward the upper right quadrant. Login to comment
135 The relative expression levels for the various mutants did not show any consistent trend; for example, the dot plot on Figure 2a suggests higher expression of cysteine-less protein, compared to G346C protein whereas the immuno-blot in Figure 1a indicates the opposite. Login to comment
138 The striking observation from the data in Figure 2b was that only one of the mutations caused a significant reduction in the transport function of P-gp, namely, isoform G346C. Login to comment
164 Figure 4 demonstrates a representative curve for the ATPase activity of purified cysteine-less P-gp and the G346C isoform in the absence or presence of the modulator nicardipine (30 µM). Login to comment
170 The most dramatic effects on ATPase activity were observed with the G346C isoform (bold, italic text), which is consistent with the altered rhodamine123 transport data (Figure 2). Login to comment
172 (a) Quadrant analysis for the cysteine-less and G346C isoforms obtained from the flow cytometry assay. Login to comment
177 Panel 3 was obtained for the G346C isoform in the absence of inhibitor. Login to comment
183 approximately 9-fold to 0.056 ( 0.007 µmol min-1 mg-1 by the G346C mutation. Login to comment
185 In contrast, unlike the control cysteine-less P-gp, the ATPase activity of the G346C mutant was not stimulated by the presence of 30 µM vinblastine. Login to comment
188 A348C also showed increased Vmax in the presence of vinblastine, but the overall fold stimulation for both substrates was comparable to that of cysteine-less P-gp. Overall, the Km of ATP was not changed for most P-gp isoforms, but for G346C and Q347C, it was reduced in the basal state when compared to that of cysteine-less P-gp. Login to comment
196 In contrast, mutations G346C, Q347C, and S349C abrogated the stimulation of ATP hydrolysis by vinblastine. Login to comment
199 The SDS-PAGE gel (8%) was silver-stained and demonstrates the retention of P-gp (G346C) on the column through the washing steps (up to 30 mM imidazole) and its elution in 120 mM imidazole. Login to comment
201 (b) Following reconstitution, an aliquot of P-gp (G346C) was subjected to sucrose density centrifugation. Login to comment
206 FIGURE 4: ATPase activity of purified, reconstituted cysteine-less and G346C isoforms of P-gp. Login to comment
207 ATPase activity was determined as a function of ATP concentration for cysteine-less P-gp (O, b) and the G346C mutant (0, 9) in the absence (open symbols) or presence (closed symbols) of 30 µM nicardipine. Login to comment
224 Consequently, the lack of any effect of vinblastine on ATPase activity in Q347C, S349C, and in particular G346C (Tables 2 and 3) is not due to major disruption of the drug binding site for this substrate by the TM6 mutation. Login to comment
225 Nucleotide Binding Characteristics of G346C. Login to comment
227 To further examine the impact of the G346C mutation on P-gp function, the binding of nucleotide was examined using the photoactive ATP analogue, [γ-32 P]-8-azido-ATP. Login to comment
228 Figure 6a demonstrates the dose-dependent binding of [γ-32 P]-8-azido-ATP to the cysteine-less and G346C isoforms of P-gp. Login to comment
229 Table 2: Michaelis-Menten Parameters for ATPase Activity of P-gpa basal nicardipine vinblastine Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) Km (mM) CYS- 0.48 ( 0.10 0.54 ( 0.05 1.37 ( 0.19 0.38 ( 0.03 0.98 ( 0.10 0.38 ( 0.02 S344C 0.30 ( 0.05 0.34 ( 0.05 1.71 ( 0.28 0.45 ( 0.07 0.84 ( 0.09 0.28 ( 0.03 V345C 0.43 ( 0.07 0.42 ( 0.06 1.69 ( 0.29 0.24 ( 0.01 0.82 ( 0.15 0.36 ( 0.04 G346C 0.06 ( 0.01* 0.21 ( 0.05* 0.15 ( 0.02* 0.24 ( 0.05 0.06 ( 0.02* 0.26 ( 0.09 Q347C 0.25 ( 0.03 0.21 ( 0.03* 0.47 ( 0.06* 0.13 ( 0.01* 0.39 ( 0.13 0.19 ( 0.02 A348C 0.79 ( 0.15 0.37 ( 0.03 2.90 ( 0.52* 0.40 ( 0.05 1.58 ( 0.30* 0.41 ( 0.06 S349C 0.38 ( 0.04 0.36 ( 0.06 1.00 ( 0.10 0.23 ( 0.03 0.45 ( 0.04 0.27 ( 0.03 A354C 0.47 ( 0.10 0.50 ( 0.10 2.21 ( 0.37* 0.59 ( 0.08* 1.29 ( 0.23 0.61 ( 0.15* G360C 0.35 ( 0.03 0.36 ( 0.02 1.88 ( 0.12 0.46 ( 0.08 1.00 ( 0.07 0.43 ( 0.02 a ATPase activity was plotted as a function of ATP concentration and the Vmax and Km parameters obtained by nonlinear regression of the Michaelis-Menten equation. Login to comment
231 Table 3: Potency and Degree of Drug Stimulation of ATP Hydrolysis by P-gpa nicardipine vinblastine EC50 (µM) fold-stimulation EC50 (µM) fold-stimulation CYS3.2 ( 0.3 3.4 ( 0.3 4.2 ( 0.7 2.4 ( 0.2 S344C 5.4 ( 0.3 5.9 ( 0.4* 12.2 ( 0.5* 2.9 ( 0.2 V345C 3.2 ( 0.1 3.9 ( 0.1 9.3 ( 1.1* 2.1 ( 0.1 G346C 5.5 ( 1.1 3.4 ( 0.3 ND 1.0 ( 0.1* Q347C 2.0 ( 0.6 2.0 ( 0.1* ND 1.3 ( 0.1* A348C 3.4 ( 0.4 3.9 ( 0.3 9.0 ( 2.1* 2.3 ( 0.2 S349C 2.3 ( 0.1 2.6 ( 0.1 ND 1.2 ( 0.1* A354C 3.5 ( 0.2 5.0 ( 0.3* 6.6 ( 0.5 2.5 ( 0.2 G360C 4.8 ( 0.5 5.5 ( 0.3* 5.9 ( 0.4 2.7 ( 0.1 a ATPase activity was plotted as a function of drug concentration and the potency and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation. Login to comment
238 Table 4: Displacement of [125 I]-Iodo-aryl-azido-prazosin Binding to P-gp Isoformsa mutant nicardipine (30 µM) vinblastine (100 µM) rhodamine123 (100 µM) hoechst33342 (100 µM) CYS- 0.36 ( 0.06 0.38 ( 0.06 1.29 ( 0.34 0.27 ( 0.05 S344C 0.48 ( 0.03 0.40 ( 0.02 1.61 ( 0.47 0.12 ( 0.01 G346C 0.41 ( 0.06 0.30 ( 0.03 1.54 ( 0.29 0.16 ( 0.05 Q347C 0.56 ( 0.10 0.45 ( 0.10 1.27 ( 0.16 0.16 ( 0.09 A348C 0.40 ( 0.03 0.36 ( 0.06 1.25 ( 0.18 0.20 ( 0.04 S349C 0.39 ( 0.05 0.34 ( 0.05 2.18 ( 0.62 0.31 ( 0.13 A354C 0.43 ( 0.04 0.39 ( 0.07 1.39 ( 0.25 0.21 ( 0.06 G360C 0.52 ( 0.12 0.34 ( 0.01 1.40 ( 1.37 0.23 ( 0.10 a The fraction of [125 I]-IAAP labeled P-gp isoforms was determined in the presence of drug and was expressed as a proportion of the amount in the absence of drug. Login to comment
241 However, the results do indicate that at a concentration of 100 µM, there was no discernible difference in the binding of the ATP analogue to cysteine-less and G346C isoforms of P-gp. Login to comment
242 To confirm that the binding of nucleotide was unaffected by the G346C mutation, the ability of ATP to displace [γ-32P]-8-azido-ATP (100 µM) photolabeling was assessed. Login to comment
243 Figure 6b shows a representative autoradiogram for the dose-dependent displacement of [γ-32 P]-8-azido-ATP binding by ATP for G346C. Login to comment
244 The potency of displacement by ATP for the control cysteine-less isoform (IC50 ) 0.37 ( 0.03 mM) was not significantly different from that observed with the G346C (IC50 ) 0.21 ( 0.12 mM) isoform, suggesting that the affinity for ATP binding is likely to be similar. Login to comment
248 An increase in the potency of displacement of [γ-32 P]-8-azido-ATP binding by ADP in G346C would signify greater affinity of the dinucleotide, thereby suggesting a slower rate of release. Login to comment
249 Figure 6c demonstrates that the ADP-mediated displacement of [γ-32 P]-8-azido-ATP binding displayed similar potencies for the cysteine-less (IC50 ) 67 ( 16 µM) and G346C (IC50 ) 72 ( 12 µM) isoforms of the protein. Login to comment
250 In summary, the impaired drug transport rate displayed by the G346C mutant isoform of P-gp is characterized by a reduced basal ATPase activity and an altered communication between the TMDs and NBDs subsequent to vinblastine, but not nicardipine, binding. Login to comment
252 Homology Modeling of TM6 in P-gp Suggests that G346C Disrupts the TMD R-Helical Packing. Login to comment
253 To provide a possible structural explanation for the unexpected functional effects of the G346C mutation, we have analyzed the native and G346C P-gp homology models, which map over 90% of the P-gp primary sequence, including TM6 in its entirety and the TMD-NBD and NBD-NBD interfaces. Login to comment
255 The backbone root-mean-square deviation (RMSD) between Sav1866 and the native and G346C P-gp models was 0.98 Å, while the RMSD between the two P-gp homology models was <0.01 Å, indicating that the backbone conformations are highly similar. Login to comment
262 The introduction of an in silico G346C mutation in the P-gp homology model shows that the 346C side chain adopts an energetically favorable conformation when it is coordinated by A342 (TM6), F303, FIGURE 6: Nucleotide binding properties of purified, reconstituted cysteine-less and G346C P-gp. Login to comment
263 (a) The autoradiogram shows the amount of [γ-32P]-8-azido-ATP bound to purified, reconstituted cysteine-less and G346C isoforms of P-gp. Login to comment
266 (b) Purified, reconstituted cysteine-less and G346C isoforms of P-gp were labeled with [γ-32P]-8-azido-ATP (100 µM) in the presence or absence of varying concentrations of ATP. Login to comment
269 (c) Purified, reconstituted cysteine-less and G346C isoforms of P-gp were labeled with [γ-32P]-8-azido-ATP (10 µM) in the presence or absence of varying concentrations of ADP. Login to comment
286 For example, the modulator nicardipine stimulated hydrolysis to an identical extent and at a similar potency in the G346C isoform compared that in the cysteine-less isoform. Login to comment
292 This accounts for the unaffected nicardipine stimulation, reduced vinblastine stimulation, and yet unaffected vinblastine binding to G346C mutant. Login to comment
301 (b) Front view of the N-terminal half of the mutant P-gp model shows that the G346C side chain (CPK spacefill) is closely coordinated by one residue from TM6, A342 (red liquorice), and two residues from TM5, F303 (lower) and I306 (upper red liquorice). Login to comment
302 A342 and I306 sterically constrain G346C, while hydrogen bonds are formed between G346C and F303. Login to comment
307 The fact that the primary effect of the G346C mutation is a reduction in basal ATP hydrolysis appears counter-intuitive since the mutated residue lies within the TMD, while ATP hydrolysis occurs at the NBDs. Login to comment
310 As shown in Figure 8, ATP hydrolysis comprises multiple discrete steps and one, or more, will be affected by the G346C mutation. Login to comment
316 The signature motif coordination of NBDs may be the rate-limiting step of the catalytic cycle, but how does the G346C mutation affect it, and is it required for basal ATP hydrolysis? Login to comment
322 The G346C mutation prevents conformational changes within the TMD such that basal route B occurs at a slower rate or alternatively, prevents step (ii) from occurring, and the protein proceeds via sub-basal route A. Login to comment
323 In addition, the G346C mutation prevents step (iia) (drug-stimulated ATP hydrolysis) from occurring for vinblastine but not nicardipine. Login to comment
325 In order to provide a theoretical visualization (as opposed to a structure-based interpretation, which remains impossible) of the G346C mutation, we have constructed a P-gp homology model based upon the structure of Sav1866. Login to comment
326 The structure-based comparison of the X-ray crystallographic data for Sav1866 (33) with the 8 Å electron microscopy data for P-gp (30) demonstrates that several of the long R-helices in Sav1866 can be superimposed onto long rod-shaped EM densities observed by electron microscopy of 2D P-gp FIGURE 8: Catalytic cycle for P-gp, the influence of the G346C mutation. Login to comment
336 Moreover, the regulation may be perturbed by the G346C mutation in TM6, suggesting a key role for this region of the protein for both the basal- and drug-stimulated activity. Login to comment
337 The rate-limiting step in ATP hydrolysis by P-gp (and other ABC transporters) is assumed to be the signature motif coordination of NBDs, and the G346C mutation in TM6 may retard the rate of this step. Login to comment

[hide] Cytosolic region of TM6 in P-glycoprotein: topogra... Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28. Storm J, Modok S, O'Mara ML, Tieleman DP, Kerr ID, Callaghan R
Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling.
Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28., 2008-03-25 [PMID:18303860]

Abstract [show]
Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.

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52 Single cysteine containing mutant isoforms of ABCB1 (S344C, V345C, G346C, Q347C, S349C, A354C, and G360C) were constructed as previously described (44) using site directed mutagenesis with the Altered Sites II (Promega) or the QuickChange (Stratagene) systems. Login to comment
117 In contrast, labeling of G346C with FM did not reach a significant extent (Lext ) 7%), even after a 300 min incubation (Figure 1a,b). Login to comment
135 (a) The V345C (upper gel) and G346C (lower gel) isoforms were reacted with CM and FM, respectively, for 10-300 min as described in Materials and Methods. Login to comment
139 Filled circles represent the labeling of V345C with CM, open circles represent the labeling of G346C with FM. Login to comment
158 The data in Table 2 confirms previous findings (44) that the G346C and Q347C mutations provided the greatest impairment to ABCB1 activity with the underlying defect caused by impaired basal activity. Login to comment
166 A more dramatic effect was observed in this isoform following CM modification, namely, that vinblastine could no longer stimulate the basal ATPase Table 1: Summary of Relative Accessibilities of TM6 Residuesa ABCB1 isoform catalytic intermediate FM BM CM S344C basal - +++ +++ AMPPNP + +++ +++ vanadate + +++ +++ V345C basal - + +++ AMPPNP + ++ +++ vanadate + + +++ G346C basal - + ++ AMPPNP - ++ ++ vanadate + + ++ Q347C basal + + +++ AMPPNP + + ++ vanadate + + +++ S349C basal - + + AMPPNP + ++ + vanadate - ++ +++ A354C basal + +++ +++ AMPPNP ++ +++ +++ vanadate + + +++ G360C basal +++ +++ ++ AMPPNP +++ +++ ++ vanadate + ++ +++ a The accessibility of each introduced cysteine residue was determined using fluorescein-maleimide (FM), BODIPY-maleimide (BM) or coumarin-maleimide (CM). Login to comment
170 Table 2: Effects of Covalent Modification by CM on ATPase Activity of Mutant ABCB1 TM6 Isoformsa basal Vmax (nmol Pi min-1 mg-1) stimulated Vmax (nmol Pi min-1 mg-1) (-)CM (+) CM % change (-)CM (+) CM % change S344C 188 ( 62 192 ( 56 857 ( 216 317 ( 93* -63 V345C 563 ( 85 388 ( 41 -31 1173 ( 355 937 ( 292 -20 G346C 101 ( 18 102 ( 12 268 ( 47 186 ( 59 -30 Q347C 66 ( 6 41 ( 15 -37 161 ( 33 61 ( 4* -62 A354C 126 ( 10 229 ( 27* +81 535 ( 72 353 ( 18* -34 G360C 396 ( 82 508 ( 134 +28 1244 ( 252 810 ( 108 -34 a The ABCB1 isoforms containing mutations within TM6 were examined for ATPase activity prior to and following reaction with the hydrophobic maleimide probe CM. Login to comment
175 Table 3: Effects of Coumarin Labeling on the Potency of Drugs to Stimulate ATP Hydrolysis by Mutant ABCB1 TM6 Isoformsa EC50 nicardipine (µM) EC50 vinblastine (µM) (-) CM (+) CM (-) CM (+) CM S344C 3.0 ( 0.4 0.5 ( 0.1* 12.1 ( 0.5 na V345C 2.3 ( 0.4 3.9 ( 0.6 4.9 ( 1.4 8.5 ( 1.9 G346C 4.8 ( 1.8 7.1 ( 1.7 na na Q347C 2.7 ( 0.4 1.9 ( 0.8 na na A354C 2.4 ( 0.2 1.6 ( 0.2 2.5 ( 0.2 na G360C 2.7 ( 0.4 5.1 ( 0.2* 5.7 ( 0.1 8.6 ( 1.2 a The potency of nicardipine and vinblastine to stimulate ATP hydrolysis was examined for a wide range of drug concentrations (10-9-10-4 M). Login to comment
186 While the G346C isoform was unaffected by the reaction with CM, the Vmax for nicardipine stimulated ATP hydrolysis was reduced 2-fold in the Q347C isoform (Table 2). Login to comment

[hide] Transmembrane helix 12 modulates progression of th... Biochemistry. 2009 Jul 7;48(26):6249-58. Crowley E, O'Mara ML, Reynolds C, Tieleman DP, Storm J, Kerr ID, Callaghan R
Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1.
Biochemistry. 2009 Jul 7;48(26):6249-58., 2009-07-07 [PMID:19456124]

Abstract [show]
Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [gamma-(32)P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.

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219 A similar study with TM6 revealed significantly fewer positions at which site-directed mutagenesis to cysteine caused functional perturbation in the basal state (notably mutant isoform G346C). Login to comment
241 In contrast, mutation of the equivalent residue (G346C) in TM6 was implicated in communication between the vinblastinebinding siteand the NBDs(37).The datacontinuetosupport our assertion that drugs communicate to the NBDs via distinct pathways and that allosteric communication between TMD1 and TMD2 to NBD1 and NBD2 is not symmetrical in ABCB1. Login to comment

[hide] Drug-stimulated ATPase activity of human P-glycopr... J Biol Chem. 1997 Aug 22;272(34):20986-9. Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
J Biol Chem. 1997 Aug 22;272(34):20986-9., 1997-08-22 [PMID:9261097]

Abstract [show]
Transmembrane segments (TM) 6 and 12 are directly connected to the ATP-binding domain in each homologous half of P-glycoprotein and are postulated to be important for drug-protein interactions. Cysteines introduced into TM6 (L332C, F343C, G346C, and P350C) were oxidatively cross-linked to cysteines introduced into TM12 (L975C, M986C, G989C, and S993C, respectively). The pattern of cross-linking was consistent with a left-handed coiled coil arrangement of the two helices. To detect conformational changes between the helices during drug-stimulated ATPase activity, we tested the effects of substrates and ATP on cross-linking. Cyclosporin A, verapamil, vinblastine, and colchicine inhibited cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C. By contrast, ATP promoted cross-linking between only L332C/L975C. Enhanced cross-linking between L332C/L975C was due to ATP hydrolysis, since cross-linked product was not observed in the presence of ATP and vanadate, ADP, ADP and vanadate, or AMP-PNP. Cross-linking between P350C/S993C inhibited verapamil-stimulated ATPase activity by about 75%. Drug-stimulated ATPase activity, however, was fully restored in the presence of dithiothreitol. These results show that TM6 and TM12 undergo different conformational changes upon drug binding or during ATP hydrolysis, and that movement between these two helices is essential for drug-stimulated ATPase activity.

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68 To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
76 Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment
100 The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine. Login to comment
105 By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment
108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein. Login to comment
109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment
126 Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP. Login to comment
132 Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 ␮M cyclosporin A, or 5 mM colchicine. Login to comment
144 Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. Login to comment

[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]

Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.

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83 There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%). Login to comment
86 A similar pattern was observed for mutants G346C, A985C, G989C, and Q990C, suggesting that the low ATPase activity in these mutants was not due to a processing defect. Login to comment
96 Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B). Login to comment
107 The inhibition of mutants G346C and G989C were not determined (ND) due to their low activities. Login to comment
141 Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment

[hide] A new structural model for P-glycoprotein. J Membr Biol. 1998 Nov 15;166(2):133-47. Jones PM, George AM
A new structural model for P-glycoprotein.
J Membr Biol. 1998 Nov 15;166(2):133-47., 1998-11-15 [PMID:9841738]

Abstract [show]
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.

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204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules. Login to comment

[hide] On the origin of large flexibility of P-glycoprote... J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8. Wen PC, Verhalen B, Wilkens S, Mchaourab HS, Tajkhorshid E
On the origin of large flexibility of P-glycoprotein in the inward-facing state.
J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8., [PMID:23658020]

Abstract [show]
P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 A, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.

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137 In addition, the G346C mutation in human Pgp (equivalent to Gly-342 of mouse Pgp, which causes a kink in helix TM6, supplemental Fig. S6) is FIGURE 4. Login to comment