Structural Insights into PLCε: Discovery of an Integrated RA1 Domain and Novel Regulatory Elements

Phospholipase C (PLC) ε enzymes hydrolyze phosphatidylinositol lipids at cellular membranes to produce inositol phosphates (IPx) and diacylglycerol. These second messengers increase intracellular Ca2+ and activate protein kinase C, allowing PLCε to contribute to numerous processes in response to G protein‐coupled receptor and receptor tyrosine kinase activation. However, aberrant PLCε activity leads to cardiac hypertrophy and upregulation of oncogenic and inflammatory signaling pathways. PLCε shares a conserved core with other PLC enzymes, but contains additional regulatory domains at its N‐ and C‐termini that contribute to autoregulation, membrane association, and G protein‐dependent activation. However, the large size and conformational heterogeneity of this enzyme present formidable hurdles for high‐resolution structure determination. Here, we present the 2.7 Å structure of a catalytically active PLCε variant, spanning the EF3‐RA1 domains, providing the first structural insights into this subfamily. This structure reveals that the C‐terminal Ras association (RA) 1 domain forms extensive intramolecular interactions with the rest of the lipase. Using biochemical and cell‐based assays, we also identified two PLCε regulatory elements: a highly conserved amphipathic helix within the catalytic domain, and the linker connecting the RA1 domain to the core. We are now pursuing high‐resolution structures of larger fragments of PLCε using domain‐specific Fabs, with the goal of using structure‐guided drug discovery to identify modulators of this critical enzyme.