ABCMdb

PMID: 12820887

Gabriel MP, Storm J, Rothnie A, Taylor AM, Linton KJ, Kerr ID, Callaghan R
Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508.
Biochemistry. 2003 Jul 1;42(25):7780-9., 2003-07-01 [PubMed]

Sentences

No. Mutations Sentence Comment

70 ABCB1 p.Ser452Cys ABCB1 p.Met450Cys ABCB1 p.Glu393Cys ABCB1 p.Arg395Cys In each case, the primer introduced the desired cysteine residue (underlined) and also a silent, Table 1: Oligonucleotide Sequences Employed to Introduce Single Cysteine Mutations into Pgpa mutation restriction site sequence (5'-3') E393C -EcoRI GA AAT TTG tgt TTC AGA AAT R395C -EcoRI TTG GAg TTC tGt AAT GTT CAC M450C +HinCII GAG GGG tgt GTC AGT GTT GAc GGA CAG S452C +HinCII GGG ATG GTC tGT GTT GAc GGA CAG a Lowercase is used to denote mutated bases, while underlined triplets denote introduced codons for cysteine. Login to comment 109 ABCB1 p.Asn508Cys The time course of [3H]-NEM association with the N508C Pgp was determined by incubating protein (250 ng) for 1-240 min (20 °C) with [3H]-NEM (1 µM). Login to comment 111 ABCB1 p.Asn508Cys To determine the effect of nucleotide on accessibility of N508C Pgp to covalent modification the protein (250 ng) was incubated with the non-hydrolysable ATP analogue AMP-PNP (0-10 mM) in buffer comprising 100 mM MOPS pH 6.8 and 5 mM MgCl2. Login to comment 114 ABCB1 p.Asn508Cys Labeling efficiency of the N508C Pgp trapped in a posthydrolytic conformation was tested following a vanadate trapping procedure based on previously published methods (12). Login to comment 118 ABCB1 p.Asn508Cys The photoaffinity labeling of wild type and N508C Pgp was based on previously published methods (12-14). Login to comment 123 ABCB1 p.Asn508Cys To assess the effect of NEM, the wild type or N508C isoforms were incubated with 100 µM NEM, in the dark for 1 h prior to the addition of [R-32 P]-azido-ATP. Login to comment 127 ABCB1 p.Asn508Cys The time-course for [3H]-NEM labeling of the N508C mutant Pgp isoform was fitted by a single phase association equation: where B ) fraction bound; Bmax ) maximal binding; k ) association rate constant (min-1 ); t ) time (min). Login to comment 145 ABCB1 p.Asn508Cys This parent protein is termed Cys-less, and single-cysteine isoforms derived from this are referred to as, for example, N508C, denoting that the asparagine at position 508 has been mutated to cysteine. Login to comment 147 ABCB1 p.Asp498Cys ABCB1 p.Lys515Cys ABCB1 p.Met450Cys ABCB1 p.Arg395Cys Although recombinant virus was produced for R395C, M450C, D498C, and K515C (determined by hybridization of radiolabeled anti-mdr1 oligonucleotides to DNA isolated from recombinant virus), we were unable to detect Pgp expression following insect cell infection. Login to comment 148 ABCB1 p.Arg580Cys No recombinant virus could be obtained for R580C despite several independent cotransfection experiments. Login to comment 149 ABCB1 p.Lys578Cys ABCB1 p.Ile500Cys ABCB1 p.Asn508Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys Therefore, our investigations concentrated on three mutations located on the R-helical subdomain (I500C, N508C, K578C) and two on the surface of the -sheet ABC-specific subdomain (E393C, S452C). Login to comment 151 ABCB1 p.Lys578Cys ABCB1 p.Asn508Cys Similar yields were obtained for Cys-less (105 ( 23 µg), N508C (121 ( 28 µg), K578C V ) (Vmax[S])/(Km + [S]) (1) V ) Vmin + (Vmax - Vmin)/(1 + 10(logEC50-L) ) (2) B ) Bmax(1 - exp-kt ) (3) FIGURE 1: Model of the N-terminal nucleotide binding subdomain of Pgp. Login to comment 155 ABCB1 p.Ile500Cys (118 ( 29 µg), while the amount of I500C (54 ( 22 µg) was approximately half. Login to comment 156 ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys Unfortunately, mutants E393C (5 ( 0.4 µg) and S452C (13 ( 3 µg) produced significantly lower yields despite several attempts to increase expression by producing higher titer recombinant baculovirus. Login to comment 173 ABCB1 p.Asn508Cys ABCB1 p.Ser452Cys The degree of inhibition varied from 78 ( 3% for S452C to 87 ( 1% for N508C and the IC50 values were in the range 0.55-0.8 µM. Similarly to the stimulatory drugs, the potency for XR9576 to affect ATP hydrolysis was not different in any mutant compared to Cys-less. Login to comment 175 ABCB1 p.Asn508Cys Vanadate produced between 91 ( 3% (N508C) and 99 ( 1% (Cys-less) inhibition of overall ATPase activity. Login to comment 182 ABCB1 p.Lys578Cys ABCB1 p.Ile500Cys ABCB1 p.Asn508Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys To Table 2: The Effects of Cysteine Replacement on Characteristics of ATP Hydrolysisa basal ATPase activity stimulated ATPase activity Pgp isoform n Km (mM) Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) fold stimulation Cys-less 10 0.52 ( 0.05 0.29 ( 0.06 0.37 ( 0.04 0.83 ( 0.12 2.9 ( 0.2 E393C 3 0.39 ( 0.03 0.47 ( 0.25 0.18 ( 0.01 1.41 ( 0.48 3.4 ( 0.8 S452C 4 0.39 ( 0.03 0.49 ( 0.13 0.31 ( 0.04 1.19 ( 0.37 2.4 ( 0.7 I500C 4 0.20 ( 0.03 0.24 ( 0.07 0.24 ( 0.04 0.51 ( 0.21 2.6 ( 0.2 N508C 4 0.54 ( 0.12 0.21 ( 0.09 0.38 ( 0.03 0.52 ( 0.10 3.2 ( 0.5 K578C 4 0.64 ( 0.17 0.31 ( 0.13 0.35 ( 0.05 0.54 ( 0.21 2.0 ( 0.2 a ATPase activity was measured for each mutant isoform of Pgp as described in Materials and Methods. Login to comment 188 ABCB1 p.Lys578Cys ABCB1 p.Ile500Cys ABCB1 p.Asn508Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys Table 3: The Effects of Cysteine Replacement on Drug Stimulation or Inhibition of ATP Hydrolysisa nicardipine vinblastine XR9576 vanadate Pgp isoform EC50 (µM) EC50 (µM) IC50 (µM) IC50 (µM) Cys-less 6.3 ( 0.1 4.6 ( 1.3 0.79 ( 0.27 3.4 ( 1.3 E393C 4.4 ( 1.4 10.8 ( 4.2 0.79 ( 0.16 4.6 ( 1.1 S452C 2.9 ( 0.8 8.2 ( 3.4 0.61 ( 0.24 4.9 ( 0.4 I500C 3.0 ( 0.6 5.4 ( 0.7 0.80 ( 0.19 5.1 ( 0.9 N508C 5.8 ( 0.9 6.2 ( 2.9 0.55 ( 0.15 7.4 ( 1.8 K578C 7.7 ( 0.5 12.4 ( 2.1 0.57 ( 0.11 4.3 ( 0.8 a ATPase activity was measured for each Pgp isoform in the presence of 2 mM Mg‚ATP with varying concentrations (3 nM to 100 µM) of either stimulatory drugs (nicardipine, vinblastine) or inhibitors (XR9576, vanadate). Login to comment 194 ABCB1 p.Asn508Cys Figure 2 shows data depicting the effects of covalent modification on drug stimulated ATPase activity in wild type, Cys-less and N508C Pgp. Login to comment 197 ABCB1 p.Lys578Cys ABCB1 p.Ile500Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys None of the mutants E393C (2.3 ( 2.1%), S452C (2.2 ( 1.9%), I500C (1.9 ( 1.2%), nor K578C (3.1 ( 1.1%) displayed significant inhibition of the drug stimulated ATPase activity in the presence of up to 100 µM NEM (data not shown). Login to comment 198 ABCB1 p.Asn508Cys In contrast, NEM was able to inhibit the ATPase activity of N508C characterized by a potency of IC50 ) 15 ( 3 µM. Login to comment 199 ABCB1 p.Asn508Cys These results suggest that either (i) only mutant N508C isoform is accessible to labeling with NEM or (ii) only residue 508C is involved in the catalytic process in Pgp. Login to comment 202 ABCB1 p.Asn508Cys Figure 3a demonstrates representative autoradiograms of the relative labeling of wild-type Pgp and the mutant N508C isoform with varying concentrations of radiolabel (0.01-1 µM). Login to comment 206 ABCB1 p.Lys578Cys Negligible labeling was observed for the Cys-less and the K578C mutant Pgp isoforms. Login to comment 207 ABCB1 p.Lys578Cys The inability to label mutant K578C correlates with the lack of effect of NEM on the ATPase activity of this mutant Pgp isoform. Login to comment 209 ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys The E393C and S452C mutant proteins were labeled with [3H]-NEM; however, the presence of contaminating bands at around 140 kDa precluded accurate quantitation. Login to comment 212 ABCB1 p.Asn508Cys Western immunoblotting analysis determined that these proteins were FIGURE 2: The effects of [3H]-NEM labeling on the ATPase activity of wild-type Pgp, cysteine-less Pgp (Cys-less) and the N508C mutant. Login to comment 216 ABCB1 p.Asn508Cys (a) A representative autoradiogram showing the relative labeling of purified wild type and N508C Pgp. Login to comment 219 ABCB1 p.Asn508Cys (b) Purified, reconstituted N508C (250 ng) was incubated with 1 µM [3H]-NEM for indicated times at 20 °C. (c) Quantitation of the Pgp associated band intensity was performed as described in the text and the mean graph obtained from four such experiments is plotted. Login to comment 220 ABCB1 p.Lys578Cys ABCB1 p.Ile500Cys ABCB1 p.Asn508Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys Table 4: The Ability of [3H]-NEM to Label Pgp Isoformsa Pgp isoform fraction labeledc wild type 1.00 ( 0.06 Cys-less 0.08 ( 0.05 E393C n/db S452C n/d I500C 0.29 ( 0.08 N508C 0.33 ( 0.02 K578C 0.03 ( 0.05 a The various purified Pgp (250 ng) isoforms were incubated with 1 µM [3 H]NEM for 1 h at 20 °C and then subjected to SDS-PAGE (8%) and autoradiography to determine the relative accessibilities of the introduced cysteine residues to covalent modification. Login to comment 226 ABCB1 p.Ile500Cys ABCB1 p.Asn508Cys [3 H]-NEM was able to efficiently label mutants I500C and N508C to approximately 30% of that observed for the wild type Pgp (Table 4). Login to comment 229 ABCB1 p.Asn508Cys Figure 3b shows a representative autoradiograph of the time dependent increase in [3 H]-NEM labeling of N508C Pgp. Login to comment 233 ABCB1 p.Asn508Cys The Ability of NEM to Perturb Drug Interaction with N508C-Pgp. Login to comment 234 ABCB1 p.Asn508Cys The data in Figure 2 demonstrated that treatment of mutant N508C with NEM produced a reduction in nicardipine stimulated ATPase activity. Login to comment 236 ABCB1 p.Asn508Cys To address this question the N508C protein was derivitized with 100 µM NEM and the ability of nicardipine, vinblastine, and paclitaxel to stimulate ATP hydrolysis measured. Login to comment 239 ABCB1 p.Asn508Cys This was corroborated by the unaltered extent of N508C labeling by [3H]-NEM (1 µM) in the presence of a fixed 30 µM concentration of nicardipine, vinblastine or paclitaxel (data not shown). Login to comment 240 ABCB1 p.Asn508Cys These results may indicate that the inhibition of ATP hydrolysis by NEM in N508C Pgp occurs during the catalytic cycle itself rather than perturbation of allosteric influences produced by drug binding. Login to comment 248 ABCB1 p.Asn508Cys The initial event in the catalytic cycle is binding of nucleotide and the ability of NEM treatment to affect this parameter in wild type and N508C Pgp is shown in Figure 5. Login to comment 249 ABCB1 p.Asn508Cys ABCB1 p.Asn508Cys The two proteins were labeled with 3 µM [R-32P]- azidoATP, the maximal achievable concentration of this Table 5: The Effects of NEM on the Extent and Potency of Drugs to Alter the ATPase Activity of N508C Pgpa nicardipine vinblastine paclitaxel EC50 (µM) stimulation EC50 (µM) stimulation EC50 (µM) stimulation -NEM 5.3 ( 0.3 3.3 ( 0.1 8.3 ( 1.2 1.6 ( 0.1 5.1 ( 2.1 2.3 ( 0.1 +NEM 6.4 ( 0.8 3.0 ( 0.2 6.9 ( 1.7 1.5 ( 0.1 5.3 ( 2.5 2.2 ( 0.2 a ATP hydrolysis was measured for the N508C Pgp mutant protein (250 ng) in the presence or absence of 100 µM NEM at a range of drug concentrations (3 nM to 100 µM). Login to comment 253 ABCB1 p.Asn508Cys FIGURE 4: The effect of sulfydryl modification on vinblastine stimulated and basal ATPase activity of mutant N508C. Login to comment 254 ABCB1 p.Asn508Cys The basal and drug stimulated ATPase activities were determined for N508C Pgp (250 ng) as described in the experimental procedures at different ATP concentrations (0-2 mM). Login to comment 255 ABCB1 p.Asn508Cys (a) ATPase activity of N508C Pgp in the presence (b) or absence (O) of 30 µM vinblastine. Login to comment 256 ABCB1 p.Asn508Cys (b) ATPase activity of N508C Pgp that had been preincubated with 100 µM NEM for 30 min at 20 °C. Catalytic activities were determined in the presence (9) or absence (0) of 30 µM vinblastine. Login to comment 260 ABCB1 p.Asn508Cys As demonstrated in Figure 5, both the wild type and N508C Pgp isoforms were labeled with the ATP analogue. Login to comment 262 ABCB1 p.Asn508Cys In contrast, no effect was observed for the NEM derivitised N508C mutant isoform which displayed similar intensity of photoaffinity labeling with [R-32P]-azidoATP. Login to comment 263 ABCB1 p.Asn508Cys Therefore, the inhibition of ATP hydrolysis caused by covalent attachment of NEM to the N508C isoform was not due to impaired binding of nucleotide. Login to comment 264 ABCB1 p.Asn508Cys ABCB1 p.Asn508Cys If the NEM derivitization of N508C has an inhibitory effect on ATP hydrolysis, one might expect that trapping of the N508C isoform with ADP and vanadate might exert a negative effect on the ability of NEM to label the protein. Login to comment 267 ABCB1 p.Asn508Cys In contrast, the non-hydrolyzable nucleotide analogue AMP-PNP up to 10 mM did not alter labeling of the nucleotide-binding domain in N508C-Pgp by 1 µM [3H]- NEM (Figure 6a). Login to comment 287 ABCB1 p.Asn508Cys Purified wild type and N508C Pgp isoforms were incubated with 3 µM Mg‚[32P]-azido-ATP for 20 min at 20 °C and then subjected to UV-light for 5 min at 4 °C. The extent of labeling for each isoform was compared with that following preincubation of the protein for 60 min with 100 µM NEM. Login to comment 289 ABCB1 p.Asn508Cys FIGURE 6: The efficiency of N508C labeling by [3H]-NEM during different stages of the catalytic cycle. Login to comment 290 ABCB1 p.Asn508Cys (a) N508C (250 ng) was labeled with [3H]-NEM (1 µM) in the presence of increasing concentrations of AMP-PNP and the inset shows a representative autoradiogram. Login to comment 291 ABCB1 p.Asn508Cys (b) N508C-Pgp was trapped with 2 mM Mg‚ATP and 200 µM vanadate for 20 min at 37 °C. Control (b) and trapped (O) protein was then labeled with varying concentrations of [3H]- NEM and the autoradiograms were analyzed to determine the extent of labeling at each concentration of [3H]-NEM. Login to comment 292 ABCB1 p.Lys578Cys The K578C mutant isoform did not display inhibition of ATPase activity by NEM, and this was attributed to the lack of detectable labeling by this maleimide-containing compound. Login to comment 293 ABCB1 p.Ile500Cys ABCB1 p.Ser452Cys ABCB1 p.Glu393Cys In contrast, the E393C, S452C, and I500C isoforms were labeled by NEM, yet this did not perturb the basal or drug stimulated ATPase activity. Login to comment 295 ABCB1 p.Asn508Cys However, the ATPase activity of the N508C mutant isoform was inhibited following covalent attachment of the maleimide-containing probe. Login to comment 297 ABCB1 p.Asn508Cys Therefore, a more detailed examination of individual stages of the catalytic cycle was undertaken to provide a molecular mechanism for the nature of NEM induced inhibition of the N508C isoform. Login to comment 299 ABCB1 p.Asn508Cys On first inspection, derivitization of the N508C protein with NEM caused a large decrease in the maximal velocity of drug stimulated ATP hydrolysis. Login to comment 307 ABCB1 p.Asn508Cys ABCB1 p.Asn508Cys In addition, albeit at low concentrations of nucleotide, the extent of [R32 P]-8-azido-ATP labeling of N508C Pgp was unaffected by prior interaction with NEM. Login to comment 309 ABCB1 p.Asn508Cys The demonstration that the non-hydrolyzable ATP analogue AMP-PNP did not impair labeling of N508C suggests that this residue does not become buried upon ATP binding, despite the considerable conformational changes the NBD undergoes (49-51). Login to comment 310 ABCB1 p.Asn508Cys The vanadate trapping technique (17) provides a useful tool to determine whether the inhibitory effect of NEM on the N508C Pgp isoform occurs immediately posthydrolysis of the γ-phosphate group. Login to comment 311 ABCB1 p.Asn508Cys The extent to which N508C Pgp was covalently labeled by [3H]-NEM was significantly impaired following vanadate trapping of the protein. Login to comment 313 ABCB1 p.Asn508Cys By inference, the prior covalent attachment of NEM to N508C would be expected to prevent or retard the previously reported conformational changes immediately following ATP hydrolysis (12, 13, 52). Login to comment