2/26/2023 0 Comments The note c 1 hr![]() ![]() ![]() The organization of the protein crystal lattice is unprecedented as it contains two distinct lipid-detergent micelles located at symmetry elements in the crystal. Collectively, our findings: (i) provide a structural rationale for the consensus amino acid sequence of DAG-sensitive C1 domains (ii) provide insight into the origins of DAG sensitivity and (iii) reveal how the unique hydrophilic/hydrophobic properties of the ligand-binding groove enable C1 domains to accommodate chemically diverse ligands. This advance enabled us to determine high-resolution structures of C1 bound to the endogenous agonist DAG and to each of four exogenous agonists of therapeutic interest. Herein, we have overcome these well-documented challenges 18, 19, 20 that have hindered the crystallization of extremely hydrophobic C1-ligand complexes for almost three decades. However, the structural basis of DAG recognition by the C1 domains has remained elusive, and the strategies for therapeutic agent design deployed to date all relied on modeling studies (reviewed in 16) based on the single available crystal structure of the C1 domain complexed to a ligand that does not activate PKC 17. These observations, combined with the central roles executed by PKCs in intracellular signaling established their DAG-sensing function as an attractive target for therapeutic intervention, with considerable promise in the treatment of Alzheimer’s disease 11, HIV/AIDS 12, 13, and cancer 14, 15. Shortly after their discovery, PKCs were identified as cellular receptors for tumor-promoting phorbol esters 10 that bind C1 domains in lieu of DAG. PKCs define a central DAG-sensing node in intracellular phosphoinositide signaling pathways that regulate cell growth, differentiation, apoptosis, and motility 9. These include protein phosphorylation (PKCs and PKDs 2, 3) DAG phosphorylation (DGKs 4) RacGTPase regulation (Chimaerins 5) Ras guanine nucleotide exchange factor activation (RasGRPs 6) Cdc42-mediated cytoskeletal reorganization (MRCK 7) and assembly of scaffolds that potentiate synaptic vesicle fusion and neurotransmitter release (Munc13s 8). The impressive diversity of DAG signaling output is mediated via its interactions with seven families of effector proteins that execute broad sets of regulatory functions 1. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKCδ) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Diacylglycerol (DAG) is a versatile lipid whose 1,2- sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. ![]()
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