Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein - Overexpression, purification, and role of cysteine 430

Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glycoprotein 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 K-m 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 K-d for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the K-m 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.