ABCB1 p.Arg395Cys
| Predicted by SNAP2: | A: D (63%), C: N (61%), D: D (66%), E: N (72%), F: D (71%), G: D (71%), H: N (97%), I: D (59%), K: N (97%), L: D (66%), M: D (63%), N: N (82%), P: D (75%), Q: N (87%), S: D (53%), T: N (78%), V: D (63%), W: D (75%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, S: N, 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.Arg395Cys 12820887:70:273
status: NEW147 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.
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ABCB1 p.Arg395Cys 12820887:147:44
status: NEW