697.2 - Single Particle Cryo-EM Reconstruction of an Activated Gβγ-PLCβ Complex

Isaac Fisher (Purdue University), Kaushik Muralidharan (Purdue University), Kennedy Outlaw (Purdue University), Elisabeth Garland-Kuntz (Purdue University ), Angeline Lyon (Purdue University, Purdue University )

G protein-coupled receptors (GPCRs) regulate diverse physiological processes in health and disease through activation of the heterotrimeric G protein subunits, Gα and Gβγ. G proteins in turn activate effector enzymes, such as phospholipase C β (PLCβ), to produce second messengers. The PLCβ subfamily has modest basal activity which is robustly increased by direct binding of Gαq. PLCβ1-3 are also directly activated by Gβγ. PLCβ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol-1,4,5 trisphosphate (IP3), activating protein kinase C (PKC) and increasing intracellular Ca2+. Inhibition of G protein-dependent activation of PLCβ has been shown to have therapeutic potential in multiple pathologies, including inflammation, cardiac hypertrophy, opioid analgesia, and cancer. Much has been learned about how Gαq binds to and activates PLCβ from structural and functional studies, but there is no consensus as to the binding site for Gβγ on PLCβ, or its mechanism of activation. Gαq and Gβγ synergistically activate PLCβ, suggesting the G proteins bind to nonoverlapping sites on the lipase and activate through independent mechanisms. Atomic resolution structures of the Gβγ-PLCβ complex are needed to fully understand the mechanism of Gβγ activation of PLCβ. We have isolated a stable, active Gβγ–PLCβ3 complex and are using single particle cryo-electron microscopy (cryo-EM) to investigate its structure. We have so far identified two particle populations using heterogeneous refinement, and generated reconstructions at 4 and 7 Å resolution. The structures reveal strong density for Gβγ binding to PLCβ through multivalent interactions with its pleckstrin homology (PH) domain, EF hands, and C2 domain, dispelling controversies about the Gβγ binding site that have persisted for over three decades. The greatest difference between structures is the position of Gβγ with respect to the PH-EF hand interface. Surprisingly, one conformation of the Gβγ–PLCβ complex is compatible with simultaneous binding of Gαi, which has not been reported in any other Gβγ–effector enzyme structure. This suggests that Gβγ may also function as a scaffold for Gαi and PLCβ, in which a pre-activated complex is maintained at the membrane. Dissociation of activated Gαi would allow rotation of Gβγ, leading to full engagement of PLCβ and maximum activation. Further investigation into the scaffolding and activating roles of Gβγ in PLCβ activation will be essential in developing chemical probes to selectively target this pathway.

Support or Funding Information

NIH NHLBI: 1RO1HL14076-01

Purdue University Center for Cancer Research, P30CA023168

lt;pgt;NIH NHLBI: 1RO1HL14076-01lt;/pgt;lt;pgt;Purdue University Center for Cancer Research, P30CA023168lt;/pgt;