Molecular modeling and site-directed mutagenesis studies of a phorbol ester-binding site in protein kinase C

The protein kinase C (PKC) binding site used by PKC activators such as phorbol esters and diacylglycerols (DAGs) has been characterized by means of molecular modeling and site-directed mutagenesis studies. Based upon a NMR-determined solution structure of the second cysteine-rich domain of PKC alpha, molecular modeling was used to study the structures of the complexes formed between the PKC receptor and a number of PKC ligands, phorbol esters, and DAGs. Site-directed mutagenesis studies identified a number of residues important to the binding of phorbol esters to PKC. Analysis of the molecular modeling and mutagenesis results allows the development of a binding model for PKC ligands for which the precise binding nature is defined. The calculated hydrogen bond energies between the protein and various ligands in this binding model are consistent with their measured binding affinities. The binding site for phorbol esters and DAGs is located in a highly conserved, hydrophobic loop region formed by residues 6-12 and 20-27. For the binding elements in phorbol esters, the oxygen at C-20 contributes most to the overall binding energy, and that at C-3 plays a significant role. The oxygen atom at C-12 is not directly involved in the interaction between phorbol esters and PKC. Our results also suggest that the oxygens at C-9 and C-13 are involved in PKC binding, while the oxygen at C-4 is of minimal significance. These results are consistent with known structure-activity relationships in the phorbol ester family of compounds. Comparisons with the X-ray structure showed that although the X-ray data support the results for oxygens at C-3, C-12, and C-20 of phorbol esters, they suggest different roles for oxygens at C-4, C-9, and C-13 Several factors which may contribute to these discrepancies are discussed.