Probing the binding of indolactam-V to protein kinase C through site-directed mutagenesis and computational docking simulations

Protein kinase C (PKC) comprises a family of ubiquitous enzymes transducing signals by the lipophilic second messenger sn-1,2-diacylglycerol (DAG). Te leocidin and its structurally simpler congener indolactam-V (ILV) bind to P KC with high affinity. In this paper, we report our computational docking s tudies on ILV binding to PKC using an automatic docking computer program, M CDOCK. In addition, we used site-directed mutagenesis to assess the quantit ative contribution of crucial residues around the binding site of PKC to th e binding affinity of ILV to PKC. On the basis of the docking studies, ILV binds to PKC in its cis-twist conformation and forms a number of optimal hy drogen bond interactions. In addition, the hydrophobic groups in ILV form " specific" hydrophobic interactions with side chains of a number of conserve d hydrophobic residues in PKC. The predicted binding mode for ILV is entire ly consistent with known structure-activity relationships and with our muta tional analysis. Our mutational analysis establishes the quantitative contr ibutions of a number of conserved residues to the binding of PKC to ILV. Ta ken together, our computational docking simulations and analysis by site-di rected mutagenesis provide a clear understanding of the interaction between ILV and PKC and the structural basis for design of novel, high-affinity, a nd isozyme-selective PKC ligands.