Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-? Exposes a G?? Protein Binding Site.

ABSTRACT: Mammalian phospholipase C-? (PLC-?) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how G?q and Rac1 bind PLC-?, there is a lack of consensus regarding the G?? binding site in PLC-?. Using FRET between cerulean fluorescent protein-labeled G?? and the Alexa Fluor 594-labeled PLC-? pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal G?? binding region in PLC-?3. We show that the isolated PH domain can compete with full-length PLC-?3 for binding G?? but not G?q, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-? PH domain as the G?? binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-?, necessitating conformational rearrangement to allow G?? binding. Blocking PH domain motion in PLC-? by cross-linking it to the EF hand domain inhibits stimulation by G?? without altering basal activity or G?q response. The fraction of PLC-? cross-linked is proportional to the fractional loss of G?? response. Cross-linked PLC-? does not bind G?? in a FRET-based G??-PLC-? binding assay. We propose that unliganded PLC-? exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-? modulates G?? access to its binding site.

SUBMITTER: Kadamur G 

PROVIDER: S-EPMC4900283 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature