Abstract
Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography. NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity. Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP. NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide. Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the Km for MgATP. The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.
Highlights
Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glycoprotein were prepared on the basis of structure predictions
A recent approach to circumvent such problems was to overexpress in bacteria recombinant domains predicted to be soluble, in fusion with either the glutathione S-transferase or the maltose-binding protein to allow their purification by affinity chromatography: this was achieved with the C-terminal nucleotidebinding domain (NBD2) from P-glycoprotein [19, 20], or with HlyB or CFTR domains [21, 22]
Design of the N-terminal Nucleotide-binding Domain from Mouse P-glycoprotein—From P-glycoprotein models based on cDNA sequence and predictions from hydrophobicity profiles [1], both N-terminal (NBD1) and C-terminal (NBD2) nucleotide-binding domains are assumed to be extrinsic with limited membrane interactions (Fig. 1, top scheme)
Summary
Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr P-glycoprotein were prepared on the basis of structure predictions. The ATPase activity and related drug transport of P-glycoprotein require both functional nucleotide-binding sites [11, 12] and are sensitive to the cysteine-specific modifier N-ethylmaleimide (NEM) [13,14,15,16,17]. A recent approach to circumvent such problems was to overexpress in bacteria recombinant domains predicted to be soluble, in fusion with either the glutathione S-transferase or the maltose-binding protein to allow their purification by affinity chromatography: this was achieved with the C-terminal nucleotidebinding domain (NBD2) from P-glycoprotein [19, 20], or with HlyB or CFTR domains [21, 22]. An alternative was to use a hexahistidine tag for fusion, in order to increase protein solubility and allow its purification by nickel-chelate chromatography
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