Functional characterization of the nucleotide binding domain of the Cryptosporidium parvum CpABC4 transporter: an iron-sulfur cluster transporter homolog.
In a previous study, we showed that the Cryptosporidium parvum ATP half-transporter CpABC4 (cgd1_1350) transcript was up-regulated in response to drug treatment with paromomycin and cyclosporine A in an in vitro infection model. CpABC4 may be directly or indirectly involved in the metabolic interactions between host and parasite in response to drug treatment and/or be involved in the intrinsic resistance to chemotherapy. In order to characterize the catalytic site of this transporter, an extended region of the nucleotide-binding domain of CpABC4 (H6-1350NBD) was expressed and purified as an N-terminal hexahistidine-tagged protein in E. coli. The presence of a single tryptophan residue enabled the intrinsic fluorescence to be monitored in response to binding of different compounds. A dose-dependent quenching of the domain's intrinsic fluorescence was observed with its natural substrate, ATP and the fluorescent analogue TNP-ATP. A similar effect was observed with progesterone as well as the flavonoids quercetin and silibinin, previously shown to inhibit parasite development in a cell-based assay. The purified domain also exhibited ATPase activity in the nanomolar range, which further confirmed correct folding and activity of the recombinant domain. The H6-1350NBD serves as a tool to test and design stereospecific inhibitors of the catalytic site, as well as other compounds that bind elsewhere in the domain that may indirectly interact with the catalytic site of the NBD of the CpABC4 transporter.
Benitez
Alvaro J
AJ
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Arrowood
Michael J
MJ
Mead
Jan R
JR
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000913:Antibodies, Protozoan / Q000378:metabolism; D002460:Cell Line; D016785:Cryptosporidium parvum / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D005419:Flavonoids / Q000378:metabolism; D042541:Intracellular Space / Q000469:parasitology; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D034101:Sporozoites / Q000378:metabolism; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D000913:Antibodies, Protozoan; D005419:Flavonoids; D007506:Iron-Sulfur Proteins; D009711:Nucleotides; D015800:Protozoan Proteins; D011374:Progesterone