Staff Research Associate and Lab Manager Purdue University West Lafayette, Indiana, United States
Elisabeth Garland (Purdue University)| Ngango Rugema (Purdue University)| Monita Sieng (Purdue University)| Kaushik Muralidharan (Purdue University)| Isaac Fisher (Purdue University)| Amanda Everly (Purdue University)| Satchal Erramilli (University of Chicago)| Michelle Van Camp (Purdue University)| Lauren Emmerson (Purdue University)| Jasmine Reyes (Purdue University)| Anthony Kossiakoff (University of Chicago)| Angeline Lyon (Purdue University, Purdue University)
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.