Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC.

Diacylglycerol (DAG)-sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High-resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane-mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high-resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH-836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor-promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC-mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG-sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can "cage" chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.