Peptidic determinants and structural model of human NDP kinase B (Nm23-H2)bound to single-stranded DNA

Isoform B of human NDP kinase (NDPK-B) was previously identified as a trans cription factor stimulating in vitro and ex vivo the transcription of the c -myc oncogene, which involves this enzyme in carcinogenesis. We have studie d the enzymatic properties of NDPK-B in the presence of several single-stra nded oligonucleotides. We show that the oligonucleotides are competitive in hibitors of the catalytic activity indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, t he presence of a guanine at the 3'-end of several different aptamers increa ses its affinity 10-fold. To define the surface of the protein contacting t he DNA within the nucleoprotein complex, we used single nanosecond laser pu lses as the cross-linking reagent and MALDI-TOF mass spectrometry to identi fy cross-linked peptides purified from proteolytic digests of the cross-lin ked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of th e complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.