ABCMdb

ABCB1 p.Val345Cys

Predicted by SNAP2:	A: D (75%), C: D (66%), D: D (91%), E: D (91%), F: D (66%), G: D (91%), H: D (91%), I: N (82%), K: D (91%), L: N (82%), M: N (57%), N: D (91%), P: D (91%), Q: D (85%), R: D (91%), S: D (85%), T: D (85%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: 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
79 Alternatively, mutant isoforms (S344C and V345C) in pFastBac-1 were transformed into DH10Bac E. coli. 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

[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
114 Figure 1a shows a representative example for the efficiency of labeling V345C with CM in the basal conformation. Login to comment
116 The data were quantified by densitometry and expressed graphically in Figure 1b. V345C was labeled fully (Lext ) 112%) with a half-life for the reaction of t½ ) 47 min. Login to comment
118 A complete graphical representation of similar labeling time-courses of two selected isoforms (V345C and A354C) is shown in Figure 2. Login to comment
120 In contrast, the V345C isoform was highly accessible to CM, with complete labeling (Lext ) 111 ( 19% P < 0.05) observed during the course of the reaction and partially accessible to the zwitterionic BM probe (Lext ) 48 ( 6%). Login to comment
121 Isoform V345C remained nonamenable to labeling with the hydrophilic FM when converted to the nucleotide bound or vanadate trapped conformation. Login to comment
122 Trapping V345C in these two conformations also failed to impact significantly on the extent of labeling observed under basal conditions for either BM or CM (Figure 2). 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
141 The maximal extent of labeling (Lext) was ascertained for the (a) V345C and (b) A354C mutant isoforms of ABCB1 using the three maleimide containing probes, FM, BM and CM. Login to comment
163 Representative full dose-response data is shown for two isoforms (S344C and V345C) in Figure 3. 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
181 Covalent modification also impacted the ATPase activity of isoform V345C, albeit in a markedly distinct fashion to that observed with S344C (Table 2 and Figure 3c and d). Login to comment
182 The basal and nicardipine stimulated Vmax values for ATP hydrolysis by CM modified V345C were indistinguishable from that observed with untreated protein (Table 2). Login to comment
200 ATPase activity was obtained for the S344C (a and b) and V345C isoforms (c and d) of ABCB1 prior to (empty symbols) or following the reaction with CM (filled symbols). 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
249 In silico mutation of the -branched valine residue to a slightly smaller cysteine residue, while less favorable in terms of residue packing, did not have a significant impact on the interhelical interactions of the V345C/L225/I306 triad. Login to comment
250 The modeled side chain of V345C faces away from the pore in the basal state model, rendering it inaccessible to the hydrophilic FM probe and only partially accessible to the zwitterionic BM probe, in accordance with the data presented here (Figure 4a and Table 1). Login to comment
251 Analysis of the AMP-PNP state model (which exhibits a "nucleotide sandwich dimer") suggests that a set of coordinated rotational and tilt angle changes in TM4, 5, and 6 around the V345C/L225/I306 triad occurs without altering the exposure of residue 345 itself (Figure 4b). Login to comment
261 On the basis of the observations in the present investigation and from previous studies (42-44), it appears that TM6 FIGURE 4: Molecular modeling insight into the V345C and A354C mutations in ABCB1. Login to comment
263 (a) In the AMP-PNP bound state, the V345C (spacefill, CPK coloring) is inaccessible to the translocation pore. Login to comment
265 The closest point of contact between the helices is V345C, which interacts hydrophobically with Leu225 (orange) and Ile306 (green). Login to comment
266 (b) The modeled basal state shows only minor reorganizations of the V345C-L225-I306 triad, consistent with the experimental findings. Login to comment
268 (d) In the basal state, conformational changes disrupt the TM3-TM6 coiled-coil motif and break the D188-V345C hydrogen bond. Login to comment