Recombinant N-terminal Nucleotide-binding Domain from Mouse P-glycoprotein

Abstract

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, Multidrug resistance of tumor cells is often associated with overexpression of P-glycoprotein, a membrane transporter that extrudes chemotherapeutic drugs using ATP hydrolysis as energy source (1, 2). The protein is encoded by the mdr gene family comprising two members in man, mdr1 and mdr2, or three in mouse, mdr1 (or mdr1b), mdr2, and mdr3 (or mdr1a). Only mdr1, and to a lower extent mdr3, was found to convey cellular multidrug resistance; mdr3 appears to be involved in detoxification/protection processes and mdr2 in phospholipid translocation (3). The function of mdr1 P-glycoprotein in normal tissues is still questioned although its relative abundance in mouse pregnant uterus and adrenal glands (4) favors a role in steroid hormone secretion (5).Structural analysis of the P-glycoprotein sequence, composed of 1276 amino acids in mouse (6), predicts two homologous halves, each containing up to six putative membrane-spanning ␣-helices and one cytoplasmically sided nucleotide-binding domain with characteristic Walker motifs A and B (7). P-glycoprotein structural organization is typical of the ATP-binding cassette (ABC) 1 superfamily including yeast (8) and protozoan parasite (9) drug transporters, and a series of different members from eukaryotic proteins, like the cystic fibrosis gene product CFTR, to bacterial transporters (10). 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). The lack of structural data about P-glycoprotein is due to its low abundance, difficult purification and membrane character, and to the lack of a highly overexpressing system (18). 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 20), or with HlyB or CFTR domains (21,22). However, the presence of a relatively high-size fusion protein might be undesirable when studying protein/protein interactions, and its release by specific proteolytic cleavage was only partial or led to unstable nucleotide-binding domains. An alternative was to use a hexahistidine tag for fusion, in order to increase protein solubility and allow its purification by ...

Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein

“…The apparent molecular mass of the recombinant domain appeared slightly higher than the theoretical value, 21.2 kDa, as also previously observed for other hexahistidine-tagged proteins (10,22). The intrinsic fluorescence spectrum of purified H 6 -NBD2 indicated that the single tryptophan residue, at position 1106 in P-glycoprotein, exhibited a hydrophobic environment because a low wavelength for maximal emission, 328 nm, was observed upon excitation at 295 nm (Fig.…”

“…Expression of the recombinant domain was induced with 2 mM IPTG for 2 hr at 37°C. Cell lysis by French press treatment and purification of recombinant H 6 -NBD2 from the soluble fraction by a nickel-nitrilotriacetic acid affinity chromatography were performed as previously described for H 6 -NBD1 (22). The fractions eluted with 250 mM imidazole were pooled and dialyzed against 20 mM potassium phosphate, 0.5 M NaCl, 20% glycerol, 0.01% HECAMEG, at pH 6.8 (dialysis buffer).…”

“…The oligonucleotide primers were purchased from Bioprobe Systems (Montreuil-sous-Bois, France) and the nickel-nitrilotriacetic acid agarose gel was from Qiagen S.A. (Courtaboeuf, France). 2Ј(3Ј)-N-methylanthraniloyl-ATP (MANT-ATP) was obtained as described (22). ATP, HECAMEG [6-O-(N-heptylcarbamoyl)-methyl-␣-D-glucopyranoside], and IPTG (isopropyl-1-thio-␤-D-galactopyranoside) were purchased from Boehringer Mannheim.…”

“…The C-terminal NBD2 domain was obtained in fusion with either glutathione S-transferase (GST) (20) or maltose-binding protein (21), but its separation from the fusion protein gave an unstable domain with low solubility (20). The N-terminal NBD1 domain was produced as a hexahistidine-tagged protein of varying length, the shortest protein corresponding to segment 395-581 being the most soluble (22). However, an extended NBD1 domain including a tryptophan residue allowed the monitoring of a hydrophobic region, interacting with modulator steroids like RU 486, located in close proximity to the ATP-binding site (10).…”

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

“…Previous studies with Pgp (Mdr1) [33], CFTR (cystic fibrosis transmembrane conductance regulator)( [34][35][36],andJ.R. Riordan,personalcommunication)orTAP [37] have all shown that the overexpressed ATPase largely forms inclusion bodies.…”