ABCMdb

ABCB1 p.Ser349Cys

Predicted by SNAP2:	A: N (78%), C: D (75%), D: D (85%), E: D (85%), F: D (63%), G: N (66%), H: D (75%), I: D (80%), K: D (85%), L: D (80%), M: N (57%), N: D (75%), P: D (66%), Q: D (80%), R: D (85%), T: N (66%), V: D (63%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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[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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No. Sentence Comment
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
196 In contrast, mutations G346C, Q347C, and S349C abrogated the stimulation of ATP hydrolysis by vinblastine. 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
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
293 A similar conclusion was demonstrated for the Q347C and S349C isoforms (Tables 3 and 4) and was also shown for the L339C isoform (29). 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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No. Sentence Comment
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
155 CM was chosen since it labels each of the chosen residues, with the exception of S349C (data not shown). 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
213 In addition, an alteration in the potency of the stimulation, or abrogation of ATPase activity, was observed for a number of residues (S344C, V345C, S349C, A354C, and G360C). Login to comment
237 The region including V345C and S349C was relatively intransigent to covalent modification with either FM or BM. Login to comment
242 The residue at the proposed cytosolic terminus of TM6 (S349C) displayed the greatest propensity for changes in accessibility, indicating conformational flexibility in this region of the helix. Login to comment