Abstract

Activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is regulated by complex mechanisms. In order to understand small molecule inhibitor interaction we established protein supply for the different forms of CaMKII. Recombinant protein purified from E.coli is inactive due to its phosphorylation status. Dephosphorylation of this protein enabled a highly productive structural campaign as well as SPR studies during hit evaluation and expansion early in the project. In absence of Ca2+/calmodulin this protein is in an inactive conformation but it can get activated upon Ca2+/calmodulin binding. Protein supply for active CaMKII was established by two distinct approaches. Introducing a point mutation mimicking autophosphorylation of T287 results in Ca2+-independent autonomous kinase activity. The supply of this protein facilitated kinetic profiling by SPR. However, for a crystallizable active conformation a shorter protein lacking the regulatory segment was needed. Production of this was possible by fusion of a substrate peptide. The mechanistic understanding from kinetic and structural analysis using active CaMKII is exploited for rational drug design.