Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain

Abstract

Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].