Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design - Université Claude Bernard Lyon 1 Accéder directement au contenu
Article Dans Une Revue Molecular and Biochemical Parasitology Année : 2007

Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design

Résumé

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.

Domaines

Parasitologie
Fichier principal
Vignette du fichier
19-Lawton 06 modifié.pdf (6.77 Mo) Télécharger le fichier
Origine : Fichiers produits par l'(les) auteur(s)

Dates et versions

hal-02111014 , version 1 (26-04-2019)

Identifiants

Citer

Philippe Lawton, Michel Pélandakis, Anne-Françoise Pétavy, Nadia Walchshofer. Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design. Molecular and Biochemical Parasitology, 2007, 152 (1), pp.101-107. ⟨10.1016/j.molbiopara.2006.12.004⟩. ⟨hal-02111014⟩
52 Consultations
159 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More