Ohri, V. | Search bioRxiv

bioRxiv2024

RhoA Allosterically Activates Phospholipase Cε via its EF Hands

Phospholipase C{varepsilon} (PLC{varepsilon}) cleaves phosphatidylinositol lipids to increase intracellular Ca2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLC{varepsilon} is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLC{varepsilon} to initiate a pathway that protects against ischemia/reperfusion injuries, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLC{varepsilon}, showing that the G protein binds a unique insertion within the PLC{varepsilon} EF hands. Deletion of or mutations to this PLC{varepsilon} insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLC{varepsilon} allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.

biochemistry↗

bioRxiv2024

Cryo-EM Structure of Phospholipase Cϵ Defines N-terminal Domains and their Roles in Activity

Phospholipase C{varepsilon} (PLC{varepsilon}) increases intracellular Ca2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLC{varepsilon} to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 [A] reconstruction of the largest fragment of PLC{varepsilon} to date in complex with an antigen binding fragment (Fab). The structure reveals that PLC{varepsilon} contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLC{varepsilon} and the first mechanistic insights into how it engages cellular membranes for activity.

biochemistry↗