ABCB1 p.Glu393Cys
| Predicted by SNAP2: | A: D (63%), C: D (59%), D: N (78%), F: D (71%), G: D (80%), H: D (63%), I: D (66%), K: D (71%), L: D (75%), M: D (59%), N: D (71%), P: D (91%), Q: N (82%), R: D (71%), S: D (53%), T: N (82%), V: D (59%), W: D (75%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[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
70 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.
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ABCB1 p.Glu393Cys 12820887:70:233
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.Glu393Cys 12820887:149:180
status: NEW156 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.
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ABCB1 p.Glu393Cys 12820887:156:23
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.Glu393Cys 12820887:182:300
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.Glu393Cys 12820887:188:261
status: NEW197 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).
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ABCB1 p.Glu393Cys 12820887:197:20
status: NEW209 The E393C and S452C mutant proteins were labeled with [3H]-NEM; however, the presence of contaminating bands at around 140 kDa precluded accurate quantitation.
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ABCB1 p.Glu393Cys 12820887:209:4
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.Glu393Cys 12820887:220:129
status: NEW293 In contrast, the E393C, S452C, and I500C isoforms were labeled by NEM, yet this did not perturb the basal or drug stimulated ATPase activity.
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ABCB1 p.Glu393Cys 12820887:293:17
status: NEW