Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein. Strategic location of a tryptophan residue.
The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.
Baubichon-Cortay
H
H
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, UPR 412 du CNRS, Lyon, France.
Baggetto
L G
LG
Dayan
G
G
Di Pietro
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D004351:Drug Resistance / Q000235:genetics*; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D005982:Glutathione Transferase