Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to glutathione-S-transferase.
CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a structured protein by its CD spectrum. Association reactions of the recombinant NBF-2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP-, ADP- and AMP-affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled adenine nucleotides occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s-1). Our findings support the concept of a central role of NBF-2 in CFTR activity regulation acting as an allosteric switch between channel opening and closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.
Randak
C
C
Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität, München, Germany.
Roscher
A A
AA
Hadorn
H B
HB
Assfalg-Machleidt
I
I
Auerswald
E A
EA
Machleidt
W
W
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose; D014301:Trinitrobenzenes
D008565:Membrane Proteins; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D014301:Trinitrobenzenes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; D005982:Glutathione Transferase