title: Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design.
abstract: Its natural resistance to antiprotozoal chemotherapy characterizes the intestinal protozoan parasite Cryptosporidium parvum and the P-glycoprotein-related multidrug resistance proteins such as CpABC3 could be involved. In order to design and study specific inhibitors of the CpABC3 nucleotide-binding domain, a hexahistidine-tagged recombinant protein encompassing the N-terminal cytosolic NBD1 domain was overexpressed in E. coli and purified. The 45 kDa H6-NBD1 displayed intrinsic fluorescent properties consistent with the presence of two Trp residues in a hydrophobic environment. The binding of ATP and the fluorescent analogue TNP-ATP produced a dose-dependent quenching as well as progesterone and the flavone quercetin. The extrinsic fluorescence of TNP-ATP was enhanced upon binding to H6-NBD1, which was only partially displaced by the natural substrate ATP. The recombinant protein hydrolyzed ATP (K(m)=145.4+/-18.2 microM), but ADP (K(m)=4.3+/-0.6mM) and AMP (K(m)=5.4+/-1.5 microM) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (K(i)=36.6+/-4.5 microM), but quercetin and progesterone were not inhibitors, evidencing different binding sites. The recombinant C. parvum H6-NBD1 should be a valuable tool for rational drug design and will allow the discrimination between specific inhibitors of the catalytic site and molecules binding to other sites.
authors.affiliation: Université Claude-Bernard Lyon 1, ISPB, Faculté de Pharmacie, EA 3741 Lyon, France. lawton@univ-lyon1.fr
authors.forename: Philippe
authors.initials: P
authors.lastname: Lawton
authors.forename: Michel
authors.initials: M
authors.lastname: Pélandakis
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Pétavy
authors.forename: Nadia
authors.initials: N
authors.lastname: Walchshofer
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000235:genetics*; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D011374:Progesterone / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011794:Quercetin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D011374:Progesterone; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011794:Quercetin