ABCMdb

PMID: 18303860

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 [PubMed]

Sentences

No. Mutations Sentence Comment

52 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys 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 ABCB1 p.Val345Cys Figure 1a shows a representative example for the efficiency of labeling V345C with CM in the basal conformation. Login to comment 115 ABCB1 p.Gly324Cys The positive control lane refers to labeling of the G324C isoform for 300 min and was assigned a value of 100%. Login to comment 116 ABCB1 p.Val345Cys 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 117 ABCB1 p.Gly346Cys 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 118 ABCB1 p.Val345Cys ABCB1 p.Ala354Cys 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 ABCB1 p.Val345Cys 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 ABCB1 p.Val345Cys Isoform V345C remained nonamenable to labeling with the hydrophilic FM when converted to the nucleotide bound or vanadate trapped conformation. Login to comment 122 ABCB1 p.Val345Cys 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 124 ABCB1 p.Ala354Cys By contrast, isoform A354C (Figure 2) displays conformation dependent labeling of probes. Login to comment 128 ABCB1 p.Ala354Cys BM labeled the A354C isoform equally well in the basal (Lext ) 99 ( 15%) and nucleotide bound conformations (Lext ) 130 ( 11%). Login to comment 134 ABCB1 p.Ser344Cys Two striking features are evident from an examination of the data presented in the table, namely, a "polarity" of accessibility along the stretch from S344C FIGURE 1: Labeling of single cysteine isoforms of ABCB1 by maleimides. Login to comment 135 ABCB1 p.Gly346Cys ABCB1 p.Val345Cys (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 137 ABCB1 p.Gly324Cys ABCB1 p.Gly324Cys The "+ve" lane refers to labeling of the G324C isoform for 300 min (b) The SDS-PAGE data was quantified using densitometric analysis, and the values were expressed as a percentage of that obtained for the G324C isoform. Login to comment 139 ABCB1 p.Gly346Cys ABCB1 p.Val345Cys Filled circles represent the labeling of V345C with CM, open circles represent the labeling of G346C with FM. Login to comment 141 ABCB1 p.Val345Cys ABCB1 p.Ala354Cys 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 144 ABCB1 p.Gly360Cys through to G360C and a disparity in the conformation dependent changes in accessibility. Login to comment 151 ABCB1 p.Ala354Cys Similarly, large changes in accessibility are observed with A354C (Figure 2). Login to comment 155 ABCB1 p.Ser349Cys CM was chosen since it labels each of the chosen residues, with the exception of S349C (data not shown). Login to comment 158 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys 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 161 ABCB1 p.Ala354Cys While there were no effects on the basal ATPase activity for any of the mutant isoforms, except for a 2-fold increase in the basal ATPase activity of A354C following CM labeling (Table 2), there were numerous distinct effects on the maximal extent of drug stimulated ATPase activity. Login to comment 163 ABCB1 p.Ser344Cys ABCB1 p.Val345Cys Representative full dose-response data is shown for two isoforms (S344C and V345C) in Figure 3. Login to comment 164 ABCB1 p.Ser344Cys S344C displayed a significant reduction in the Vmax and degree of stimulation of ATP hydrolysis following the attachment of the coumarin moiety. Login to comment 166 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys 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 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys 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 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys 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 ABCB1 p.Ser344Cys ABCB1 p.Val345Cys 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 ABCB1 p.Val345Cys 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 186 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys 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 187 ABCB1 p.Ala354Cys A354C, which was avidly labeled by CM, displayed a both small reduction in the Vmax for nicardipine stimulation of ATPase activity due to a reduced degree of stimulation by this compound. Login to comment 189 ABCB1 p.Gly360Cys G360C was less amenable to labeling with CM and only displayed a minor reduction in the potency of nicardipine to stimulate hydrolysis. Login to comment 194 ABCB1 p.Gln347Cys ABCB1 p.Ser344Cys ABCB1 p.Ala354Cys The isoforms S344C, Q347C, and A354C displayed significant reductions in drug stimulated ATP hydrolysis following CM labeling, and this may be attributed to altered drug binding. Login to comment 200 ABCB1 p.Ser344Cys ABCB1 p.Val345Cys 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 204 ABCB1 p.Gln347Cys ABCB1 p.Gln347Cys ABCB1 p.Gln347Cys ABCB1 p.Ser344Cys ABCB1 p.Ser344Cys ABCB1 p.Ser344Cys ABCB1 p.Ala354Cys ABCB1 p.Ala354Cys ABCB1 p.Ala354Cys Table 4: Effects of Coumarin Labeling on Drug Binding in Mutant ABCB1 TM6 Isoformsa vinblastine nicardipine S344C Q347C A354C S344C Q347C A354C (-) CM 0.34 ( 0.09 0.39 ( 0.06 0.61 ( 0.11 0.35 ( 0.02 0.40 ( 0.03 0.64 ( 0.09 (+) CM 0.46 ( 0.06 0.34 ( 0.09 0.39 ( 0.21 0.27 ( 0.03 0.41 ( 0.06 0.57 ( 0.16 a [125I]-IAAP photoaffinity labeling was undertaken in the S344C, Q347C and A354C mutant TM6 isoforms prior to and following labeling with coumarin maleimide. Login to comment 212 ABCB1 p.Gln347Cys ABCB1 p.Ser344Cys ABCB1 p.Ala354Cys Following labeling, three of the residues we have studied (S344C, Q347C, and A354C) were associated with a reduction in the magnitude to which drug substrates stimulate ATP hydrolysis. Login to comment 213 ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys 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 234 ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys The observations were also undertaken for two residues (A354C and G360C) within the extension of TM6 that links the helix to the N-terminal NBD. Login to comment 237 ABCB1 p.Val345Cys ABCB1 p.Ser349Cys The region including V345C and S349C was relatively intransigent to covalent modification with either FM or BM. Login to comment 239 ABCB1 p.Phe343Cys ABCB1 p.Val331Cys This lack of change is in contrast to the considerable conformational changes previously observed in the segment between V331C to F343C (42). Login to comment 240 ABCB1 p.Ser344Cys Residue S344C, which is located in the central region of TM6, was considerably more accessible to covalent modification than the more cytosolic region of the helix. Login to comment 241 ABCB1 p.Phe343Cys This is in good agreement with the data from Rothnie et al. demonstrating similar high accessibility for the adjacent residue, F343C (42). Login to comment 242 ABCB1 p.Ser349Cys 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 249 ABCB1 p.Val345Cys 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 ABCB1 p.Val345Cys 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 ABCB1 p.Val345Cys 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 254 ABCB1 p.Ala354Cys Similar analysis helps rationalize the A354C data and provides further evidence that our homology models are a valid reflection of TM helix packing. Login to comment 256 ABCB1 p.Ala354Cys A354C forms part of the TM3-TM6 contact interface with the sulfur atom being accessible to the translocation pore, thus enabling covalent modification by the hydrophilic FM and zwitterionic BM probes. Login to comment 257 ABCB1 p.Ala354Cys Further examination of the residues adjacent to A354C revealed a number of charged and polar residues in its immediate vicinity, in particular, K181, E184, and D188 from TM3, and the adjacent E353 from TM6, creating a favorable local environment for FM and BM. Login to comment 258 ABCB1 p.Ala354Cys Comparison of the AMP-PNP state model with the basal state model shows a disruption of the TM3-TM6 interface around A354C (Figure 4c). Login to comment 259 ABCB1 p.Ala354Cys ABCB1 p.Ala354Cys In the basal conformation homology model, the side chain of A354C was angled away from the pore, while charged residues in the local vicinity faced into the translocation pore (Figure 4d), decreasing the surface accessibility of A354C to the translocation pore and thus the accessibility to FM. Login to comment 260 ABCB1 p.Ala354Cys ABCB1 p.Ala354Cys The position of A354C at the interface of TM3 and TM6, which both have a lipid exposed face, allowed the hydrophobic CM probe to access A354C through the bilayer. Login to comment 261 ABCB1 p.Val345Cys ABCB1 p.Ala354Cys 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 ABCB1 p.Val345Cys (a) In the AMP-PNP bound state, the V345C (spacefill, CPK coloring) is inaccessible to the translocation pore. Login to comment 265 ABCB1 p.Val345Cys The closest point of contact between the helices is V345C, which interacts hydrophobically with Leu225 (orange) and Ile306 (green). Login to comment 266 ABCB1 p.Val345Cys (b) The modeled basal state shows only minor reorganizations of the V345C-L225-I306 triad, consistent with the experimental findings. Login to comment 267 ABCB1 p.Ala354Cys (c) A view from the top of the pore shows the AMP-PNP configuration: A354C (orange) hydrogen bonds to its nearest neighbour, D188 (cyan). Login to comment 268 ABCB1 p.Val345Cys (d) In the basal state, conformational changes disrupt the TM3-TM6 coiled-coil motif and break the D188-V345C hydrogen bond. Login to comment 269 ABCB1 p.Ala354Cys Adjacent charged residues from TM3 (cyan) and TM6 (purple) face the translocation pore, while A354C is situated in a cleft between TM3 and TM6. Login to comment