%0 Journal Article %T Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design %+ Laboratoire de Parasitologie %+ Université Claude Bernard Lyon 1 (UCBL) %+ Amibes et interactions (A&I) %+ Molécules bioactives et chimie médicinale (B2MC) %A Lawton, Philippe %A Pélandakis, Michel %A Pétavy, Anne-Françoise %A Walchshofer, Nadia %< avec comité de lecture %@ 0166-6851 %J Molecular and Biochemical Parasitology %I Elsevier %V 152 %N 1 %P 101-107 %8 2007-03 %D 2007 %R 10.1016/j.molbiopara.2006.12.004 %K Cryptosporidium parvum %K Parasite %K Nucleotide-binding domain %K CpABC3 %K NBD1 %Z Life Sciences [q-bio]/Microbiology and Parasitology/ParasitologyJournal articles %X 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 over-expressed 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 M), but ADP (K m = 4.3 ± 0.6 mM) and AMP (K m = 5.4 ± 1.5 M) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (K i = 36.6 ± 4.5 M), 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. %G English %2 https://univ-lyon1.hal.science/hal-02111014/document %2 https://univ-lyon1.hal.science/hal-02111014/file/19-Lawton%2006%20modifi%C3%A9.pdf %L hal-02111014 %U https://univ-lyon1.hal.science/hal-02111014 %~ CNRS %~ UNIV-LYON1 %~ INSA-LYON %~ BIOENVIS %~ B2MC %~ UMR5240 %~ INSA-GROUPE %~ UDL %~ UNIV-LYON