Structural basis of binding of high-affinity ligands to protein kinase C: Prediction of the binding modes through a new molecular dynamics method andevaluation by site-directed mutagenesis

The structural basis of protein kinase C (PKC) binding to several classes o f high-affinity ligands has been investigated through complementary computa tional and experimental methods. Employing a recently developed q-jumping m olecular dynamics (MD) simulation method, which allows us to consider the f lexibility of both the ligands and the receptor in docking studies, we pred icted the binding models of phorbol-13-acetate, phorbol-12,13-dibutyrate (P DBu), indolactam V (ILV), ingenol-3-benzoate, and thymeleatoxin to PKC. The "predicted" binding model for phorbol-13-acetate is virtually identical to the experimentally determined binding model for this ligand. The predicted binding model for PDBU is the same as that for phorbol-13-acetate in terms of the hydrogen-bonding network and hydrophobic contacts. The predicted bi nding model for ILV is the same as that obtained in a previous docking stud y using a Monte Carlo method and is consistent with the structure-activity relationships for this class of ligands. Together with the X-ray structure of phorbol-13-acetate in complex with PKCG Gib, the predicted binding model s of PDBu, ILV, ingenol-3-benzoate, and thymeleatoxin in complex with PKC s howed that the binding of these ligands to PKC is governed by a combination of several highly specific and optimal hydrogen bonds and hydrophobic cont acts. However, the hydrogen-bonding network for each class of ligand is som ewhat different and the number of hydrogen bonds formed between PKC and the se ligands has no correlation with their binding affinities. To provide a d irect and quantitative assessment of the contributions of several conserved residues around the binding site to PKC-ligand binding, we have made 11 mu tations and measured the binding affinities of the high-affinity PKC ligand s to these mutants. The results obtained through site-directed mutagenic an alysis support our predicted binding models for these ligands and provide n ew insights into PKC-ligand binding. Although all the ligands have high aff inity for the wild-type PKCG Gib, our site-directed mutagenic results showe d that ILV is the ligand most sensitive to structural perturbations of the binding site while ingenol-3-benzoate is the least sensitive among the four classes of ligands examined here. Finally, we have employed conventional M D simulations to investigate the structural perturbations caused by each mu tation to further examine the role played by each individual residue in PKC -ligand binding. MD simulations revealed that several mutations, including Proll --> Gly, Leu21 --> Gly, Leu24 --> Gly, and Gln27 --> Gly, cause a rat her large conformational alteration to the PKC binding site and, in some ca ses, to the overall structure of the protein. The complete abolishment or t he significant reduction in PKC-ligand binding observed for these mutants t hus reflects the loss of certain direct contacts between the side chain of the mutated residue in PKC and ligands as well as the large conformational alteration to the binding site caused by the mutation.