| Death associated protein kinase (DAPK) is a member of the Ca2+ /calmodulin (CaM)-regulated family of serine/threonine protein kinases. The role of DAPK's kinase activity in eukaryotic cell apoptosis, and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug discovery target for neurodegenerative disorders. Kinases are considered an important class of drug targets; however, direct comparisons of protein kinase structures in the ADP-bound and AMPPNP-bound forms reflecting nucleotide product and substrate complexes, respectively, are limited. First, to gain insight into the DAPK catalytic mechanism with the broader application of insight into strategies for inhibitor design, the high resolution crystal structures of DAPK in complex with ADP, ADP-Mg 2+, AMPPNP, and AMPPNP-Mg2+ were determined. A comparative analysis of the structures revealed little change in active site residues, while a localized change in the glycine-rich loop was noted. Further, we report a potential function of Mg2+ in catalysis is to aid in conformational selection of the beta-phosphate of ADP. Second, to probe enzyme activity with structural perturbations, Gln23 within the glycine-rich loop was mutated to a CaM-regulated kinase equivalent residue (Val). The mutation of DAPKQ23 to Val resulted in a 10-fold loss in catalytic efficiency. The high resolution crystal structures of DAPKQ23V in complex with ADP were determined. These results provide a starting point and structural foundation for a molecular mechanism in which DAPK could be regulated by modulation of the glycine-rich loop. Third, we extended DAPK-nucleotide information to structure and activity investigations of the gatekeeper mutation, DAPKL93G. DAPKL93G was selectively inhibited by N6-modified adenosine analogs and the high resolution crystal structures of DAPKL93G in complex with N 6-modified adenosine analogs were determined. These results provide feasibility data for future investigations which utilize orthogonal nucleotide anchoring points for DAPK substrate discovery. |
