CALCIUM-BINDING PROPERTIES OF A CALCIUM-DEPENDENT PROTEIN-KINASE FROM PLASMODIUM-FALCIPARUM AND THE SIGNIFICANCE OF INDIVIDUAL CALCIUM-BINDING SITES FOR KINASE ACTIVATION

Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates With Ca-45(2+). Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca2+-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca2+-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca2+-induced conformational change and the Ca2+-dependent activation. Mutations at sites III and IV have minor effects.