NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD.
C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.
Chavan
A J
AJ
College of Pharmacy, Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40536.
Nemoto
Y
Y
Narumiya
S
S
Kozaki
S
S
Haley
B E
BE
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001905:Botulinum Toxins; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003014:Clostridium botulinum / Q000201:enzymology*; D008670:Metals; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*
D000345:Affinity Labels; D008670:Metals; D009243:NAD; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins