Photoaffinity labeling of diphtheria toxin fragment a with 8- azidoadenosyl nicotinamide adenine dinucleotide

Diphtheria toxin fragment A (DT-A) is an important enzyme in the class of mono(ADP-ribosyl)transferases. To identify peptides and amino acid residues which form the NAD⁺ binding site of DT-A using a photoaffinity approach, the photoprobes nicotinamide 8-azidoadenine dinucleotide (8-N³- NAD) and nicotinamide 2-azidoadenine dinucleotide (2-N³-NAD) were synthesized. Binding studies gave an IC₅₀ of 2.5μM for 8-N₃-NAD and 5.0μM for 2-N₃-NAD. Irradiation of DT-A and low concentrations of [α- ³²P]-8-N₃-NAD with short-wavelength UV light resulted in rapid covalent incorporation of the photoprobe into the protein. The photoincorporation was shown to be specific for the active site with a stoichiometry of photoincorporation of 75-80%. After proteolytic digestion of photolabeled DT- A, derivatized peptides were isolated using immobilized boronate affinity chromatography followed by reversed phase HPLC. Radiolabeled peptides originating from two regions of the protein were identified. Chymotryptic digestion produced labeled peptides corresponding to His²¹-Gln³² and Lys³³-Phe⁵³. Lys-C digestion gave overlapping peptides Ser¹¹-Lys³³ and Ser⁴⁰-Lys⁵⁹. Tyr²⁷ was identified as the site of photoinsertion within the peptide His²¹-Gln³² on the basis of the absence of PTH-Tyr at the predicted cycle during sequence analysis and by the lack of predicted chymotryptic cleavage at Tyr27. Within the second modified peptide Ser⁴⁰- Lys⁵⁹, Trp⁵⁰ is the most probable site of modification. Identification of Tyr²⁷ as a site of photoinsertion is in agreement with its placement in the NAD binding site of the X-ray structure of the proenzyme DT-NAD complex [Bell, C. E., and Eisenberg, D. (1996) Biochemistry 35, 1137]. Trp⁵⁰ is far from the adenine ring in the crystallographic model; however, site-directed mutagenesis studies suggest that Trp⁵⁰ is a major determinant of NAD binding affinity [Wilson, B. A., Blanke, S. R., Reich, K. A., and Collier, R. J. (1994) J. Biol. Chem. 269, 23296-23301].