ABCB1 p.Asn508Cys
| Predicted by SNAP2: | A: D (66%), C: D (59%), D: D (63%), E: D (66%), F: D (71%), G: D (53%), H: N (66%), I: D (71%), K: D (66%), L: D (71%), M: D (66%), P: D (80%), Q: D (53%), R: D (71%), S: D (59%), T: D (59%), V: D (71%), W: D (75%), Y: D (53%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Cysteine-scanning mutagenesis provides no evidence... EMBO J. 1999 Dec 1;18(23):6800-8. Blott EJ, Higgins CF, Linton KJ
Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.
EMBO J. 1999 Dec 1;18(23):6800-8., 1999-12-01 [PMID:10581253]
Abstract [show]
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.
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No. Sentence Comment
190 Oligonucleotides used for site-directed mutagenesis pSC-name Diagnostic restriction site Mutagenic oligonucleotide sequence 5Ј-3Ј N280C ϩAvaI GGTACAACAAATGTCTCGAGGAAGCTAAAAG G324C -AflIII CCTTGGTCTTATCATGTGAATATTCTATTGG S403C ϩPstI CTTCAGTTACCCCTGCAGAAAAGAAGTTAAG S419C ϩEco57I GAACCTGAAAGTGCAGTGTGGGCAGACG Q456C ϩBstEI GTTGATGGATGCGATATCCGGACCATAAATG F465C ϩBanI ATGTAAGGTGCCTACGGGAA I469C ϩNsiI CTACGGGAATGCATTGGTGT S474C ϩHpaII GGTGTCGTGTGTCAGGAACCGGTATTGTTT T482C ϩBsrGI GTATTGTTTGCCTGTACAATAGCTGAAAAC I488C ϩEclXI GCTGAAAACTGTCGCTACGGCCGTGAAAATG Y490C none ACATTCGCTGTGGCCGTGA V495C ϩBsrGI GGCCGTGAAAATTGTACAATGGATGAGATTG D498C -NcoI GTCACCATGTGTGAGATTGAG I500C ϩBsmI GTCACCATGGATGAATGCGAGAAAGCTGTC K502C ϩBsmI GGATGAGATTGAATGCGCTGTCAAGGAAG N508C ϩFspI GTCAAGGAAGCATGCGCATATGACTTTATC D511C ϩAflIII GGAAGCCAACGCGTATTGCTTTATCATG K515C ϩAflIII GAAGCCAACGCGTATGACTTTATCATGTGCCTGCCTCAT K519C ϩStyI GAAACTGCCTCATTGCTTTGACACCTTGGTTGGAGAGAGAG A529C ϩBanI GAGAGAGGGTGCCAGTTGAG K536C ϩBlpI GAGAGAGAGGCGCCCAGTTGAGTGGTGGGCAGTGCCAGAGGATCG R547C ϩBssSI GCACGTGCCCTCGTGTGCAACCCCAAG P549C ϩSspI TGGTTCGCAACTGCAAAATATTCCTGCTGGA L554C ϩSspI CGCAACCCCAAAATATTGCTGTGCGATGAGGCCACG S565C ϩBsmI GACACAGAATGCGAAGCAG V569C ϩBsmI GACACAGAATGCGAAGCAGTGTGTCAGGTGG K578C ϩBsiEI GATAAGGCCAGATGCGGCCGGACCACC R580C -MslI GCCACAAAAGGTTGCACGACCATTGTGATA T581C ϩApaLI GAAAAGGTCGGTGCACCATTGTG E638C ϩEclXI GAAGTTGAATTATGCAATGCGGCCGATGAATC 'ϩ` represents the introduction of a new restriction endonuclease site; '-` represents the removal of an existing site.
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ABCB1 p.Asn508Cys 10581253:190:830
status: NEW[hide] Communication between the nucleotide binding domai... Biochemistry. 2003 Jul 1;42(25):7780-9. 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 [PMID:12820887]
Abstract [show]
Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.
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No. Sentence Comment
109 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).
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ABCB1 p.Asn508Cys 12820887:109:49
status: NEW111 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.
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ABCB1 p.Asn508Cys 12820887:111:58
status: NEW114 Labeling efficiency of the N508C Pgp trapped in a posthydrolytic conformation was tested following a vanadate trapping procedure based on previously published methods (12).
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ABCB1 p.Asn508Cys 12820887:114:27
status: NEW118 The photoaffinity labeling of wild type and N508C Pgp was based on previously published methods (12-14).
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ABCB1 p.Asn508Cys 12820887:118:44
status: NEW123 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.
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ABCB1 p.Asn508Cys 12820887:123:46
status: NEW127 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).
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ABCB1 p.Asn508Cys 12820887:127:45
status: NEW145 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.
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ABCB1 p.Asn508Cys 12820887:145:120
status: NEW149 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).
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ABCB1 p.Asn508Cys 12820887:149:105
status: NEW151 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.
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ABCB1 p.Asn508Cys 12820887:151:62
status: NEW173 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.
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ABCB1 p.Asn508Cys 12820887:173:70
status: NEW175 Vanadate produced between 91 ( 3% (N508C) and 99 ( 1% (Cys-less) inhibition of overall ATPase activity.
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ABCB1 p.Asn508Cys 12820887:175:35
status: NEW182 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.
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ABCB1 p.Asn508Cys 12820887:182:498
status: NEW188 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).
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ABCB1 p.Asn508Cys 12820887:188:406
status: NEW194 Figure 2 shows data depicting the effects of covalent modification on drug stimulated ATPase activity in wild type, Cys-less and N508C Pgp.
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ABCB1 p.Asn508Cys 12820887:194:129
status: NEW198 In contrast, NEM was able to inhibit the ATPase activity of N508C characterized by a potency of IC50 ) 15 ( 3 µM.
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ABCB1 p.Asn508Cys 12820887:198:60
status: NEW199 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.
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ABCB1 p.Asn508Cys 12820887:199:50
status: NEW202 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).
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ABCB1 p.Asn508Cys 12820887:202:110
status: NEW212 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.
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ABCB1 p.Asn508Cys 12820887:212:189
status: NEW216 (a) A representative autoradiogram showing the relative labeling of purified wild type and N508C Pgp.
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ABCB1 p.Asn508Cys 12820887:216:91
status: NEW219 (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.
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ABCB1 p.Asn508Cys 12820887:219:28
status: NEW220 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.
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ABCB1 p.Asn508Cys 12820887:220:168
status: NEW226 [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).
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ABCB1 p.Asn508Cys 12820887:226:58
status: NEW229 Figure 3b shows a representative autoradiograph of the time dependent increase in [3 H]-NEM labeling of N508C Pgp.
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ABCB1 p.Asn508Cys 12820887:229:104
status: NEW233 The Ability of NEM to Perturb Drug Interaction with N508C-Pgp.
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ABCB1 p.Asn508Cys 12820887:233:52
status: NEW234 The data in Figure 2 demonstrated that treatment of mutant N508C with NEM produced a reduction in nicardipine stimulated ATPase activity.
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ABCB1 p.Asn508Cys 12820887:234:59
status: NEW236 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.
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ABCB1 p.Asn508Cys 12820887:236:29
status: NEW239 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).
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ABCB1 p.Asn508Cys 12820887:239:49
status: NEW240 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.
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ABCB1 p.Asn508Cys 12820887:240:75
status: NEW248 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.
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ABCB1 p.Asn508Cys 12820887:248:141
status: NEW249 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).
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ABCB1 p.Asn508Cys 12820887:249:204
status: NEWX
ABCB1 p.Asn508Cys 12820887:249:499
status: NEW253 FIGURE 4: The effect of sulfydryl modification on vinblastine stimulated and basal ATPase activity of mutant N508C.
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ABCB1 p.Asn508Cys 12820887:253:109
status: NEW254 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).
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ABCB1 p.Asn508Cys 12820887:254:68
status: NEW255 (a) ATPase activity of N508C Pgp in the presence (b) or absence (O) of 30 µM vinblastine.
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ABCB1 p.Asn508Cys 12820887:255:23
status: NEW256 (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.
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ABCB1 p.Asn508Cys 12820887:256:23
status: NEW260 As demonstrated in Figure 5, both the wild type and N508C Pgp isoforms were labeled with the ATP analogue.
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ABCB1 p.Asn508Cys 12820887:260:52
status: NEW262 In contrast, no effect was observed for the NEM derivitised N508C mutant isoform which displayed similar intensity of photoaffinity labeling with [R-32P]-azidoATP.
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ABCB1 p.Asn508Cys 12820887:262:60
status: NEW263 Therefore, the inhibition of ATP hydrolysis caused by covalent attachment of NEM to the N508C isoform was not due to impaired binding of nucleotide.
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ABCB1 p.Asn508Cys 12820887:263:88
status: NEW264 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.
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ABCB1 p.Asn508Cys 12820887:264:29
status: NEWX
ABCB1 p.Asn508Cys 12820887:264:117
status: NEW267 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).
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ABCB1 p.Asn508Cys 12820887:267:133
status: NEW287 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.
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ABCB1 p.Asn508Cys 12820887:287:23
status: NEW289 FIGURE 6: The efficiency of N508C labeling by [3H]-NEM during different stages of the catalytic cycle.
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ABCB1 p.Asn508Cys 12820887:289:28
status: NEW290 (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.
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ABCB1 p.Asn508Cys 12820887:290:4
status: NEW291 (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.
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ABCB1 p.Asn508Cys 12820887:291:4
status: NEW295 However, the ATPase activity of the N508C mutant isoform was inhibited following covalent attachment of the maleimide-containing probe.
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ABCB1 p.Asn508Cys 12820887:295:36
status: NEW297 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.
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ABCB1 p.Asn508Cys 12820887:297:179
status: NEW299 On first inspection, derivitization of the N508C protein with NEM caused a large decrease in the maximal velocity of drug stimulated ATP hydrolysis.
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ABCB1 p.Asn508Cys 12820887:299:43
status: NEW307 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.
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ABCB1 p.Asn508Cys 12820887:307:103
status: NEW309 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).
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ABCB1 p.Asn508Cys 12820887:309:92
status: NEW310 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.
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ABCB1 p.Asn508Cys 12820887:310:117
status: NEW311 The extent to which N508C Pgp was covalently labeled by [3H]-NEM was significantly impaired following vanadate trapping of the protein.
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ABCB1 p.Asn508Cys 12820887:311:20
status: NEW313 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).
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ABCB1 p.Asn508Cys 12820887:313:54
status: NEW