ABCMdb

PMID: 17696319

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 [PubMed]

Sentences

No. Mutations Sentence Comment

66 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys ABCB1 p.Ala348Cys 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 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys ABCB1 p.Ala348Cys 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 79 ABCB1 p.Ser344Cys ABCB1 p.Val345Cys Alternatively, mutant isoforms (S344C and V345C) in pFastBac-1 were transformed into DH10Bac E. coli. Login to comment 117 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys In addition, the data obtained for G346C displayed a shift from the lower right quadrant toward the upper right quadrant. Login to comment 135 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys (a) Quadrant analysis for the cysteine-less and G346C isoforms obtained from the flow cytometry assay. Login to comment 177 ABCB1 p.Gly346Cys Panel 3 was obtained for the G346C isoform in the absence of inhibitor. Login to comment 183 ABCB1 p.Gly346Cys approximately 9-fold to 0.056 ( 0.007 µmol min-1 mg-1 by the G346C mutation. Login to comment 185 ABCB1 p.Gly346Cys 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 187 ABCB1 p.Gln347Cys ABCB1 p.Ala354Cys ABCB1 p.Ala348Cys The Vmax in the presence of nicardipine was also reduced for Q347C but increased for the A348C and A354C isoforms. Login to comment 188 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ala348Cys 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 189 ABCB1 p.Gln347Cys ABCB1 p.Ala354Cys For Q347C, the Km remained lower in the presence of nicardipine, and in the presence of both nicardipine and vinblastine, the Km value was higher for A354C. Login to comment 194 ABCB1 p.Gln347Cys ABCB1 p.Ser344Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys It was increased for S344C, A354C, and G360C, and decreased for Q347C, but the potency of stimulation was unchanged compared to that of cysteine-less P-gp. Login to comment 196 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser349Cys In contrast, mutations G346C, Q347C, and S349C abrogated the stimulation of ATP hydrolysis by vinblastine. Login to comment 199 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys (b) Following reconstitution, an aliquot of P-gp (G346C) was subjected to sucrose density centrifugation. Login to comment 206 ABCB1 p.Gly346Cys FIGURE 4: ATPase activity of purified, reconstituted cysteine-less and G346C isoforms of P-gp. Login to comment 207 ABCB1 p.Gly346Cys 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 218 ABCB1 p.Ala348Cys Figure 5 provides representative autoradiograms for the specific photolabeling of the substrate [125 I]- IAAP to the cysteine-less and A348C isoforms of P-gp. Login to comment 224 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser349Cys 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 ABCB1 p.Gly346Cys Nucleotide Binding Characteristics of G346C. Login to comment 227 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys 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 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys ABCB1 p.Ala348Cys 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 ABCB1 p.Gln347Cys ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Ser344Cys ABCB1 p.Val345Cys ABCB1 p.Val345Cys ABCB1 p.Gly360Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys ABCB1 p.Ser349Cys ABCB1 p.Ala348Cys ABCB1 p.Ala348Cys 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 234 ABCB1 p.Ala348Cys The binding of [125I]-IAAP was measured by autoradiography of photoaffinity labeled purified, reconstituted cysteine-less and A348C isoforms of P-gp in the presence or absence of various drug substrates (100 µM). Login to comment 238 ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Ser344Cys ABCB1 p.Gly360Cys ABCB1 p.Ala354Cys ABCB1 p.Ser349Cys ABCB1 p.Ala348Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys Homology Modeling of TM6 in P-gp Suggests that G346C Disrupts the TMD R-Helical Packing. Login to comment 253 ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys (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 ABCB1 p.Gly346Cys (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 ABCB1 p.Gly346Cys (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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys This accounts for the unaffected nicardipine stimulation, reduced vinblastine stimulation, and yet unaffected vinblastine binding to G346C mutant. Login to comment 293 ABCB1 p.Gln347Cys ABCB1 p.Leu339Cys ABCB1 p.Ser349Cys 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 301 ABCB1 p.Gly346Cys (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 ABCB1 p.Gly346Cys ABCB1 p.Gly346Cys A342 and I306 sterically constrain G346C, while hydrogen bonds are formed between G346C and F303. Login to comment 307 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys In addition, the G346C mutation prevents step (iia) (drug-stimulated ATP hydrolysis) from occurring for vinblastine but not nicardipine. Login to comment 325 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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 ABCB1 p.Gly346Cys 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