Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum.
The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1). Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate]. Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.
Daniel
D
Nagore
Marta
M
Llarena
María J
MJ
Llama
Juan L
JL
Serra
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D000091285:Nitrate Transporters; D009566:Nitrates / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D027321:Anion Transport Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000091285:Nitrate Transporters; D009566:Nitrates; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate