title: Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.
abstract: Mammalian phospholipase C-β (PLC-β) 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 Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β 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-β modulates Gβγ access to its binding site.
authors.affiliation: From the Department of Pharmacology, Molecular Biophysics Graduate Program, and Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Kadamur
authors.affiliation: From the Department of Pharmacology, Molecular Biophysics Graduate Program, and Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Elliott.Ross@UTSouthwestern.edu.
authors.forename: Elliott M
authors.identifier: http://orcid.org/0000-0002-0252-8197
authors.initials: EM
authors.lastname: Ross
mesh_terms: D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D054799:Phospholipase C beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010750:Phosphoproteins; C107953:RAC1 protein, human; D011994:Recombinant Proteins; C039077:platelet protein P47; C518039:PLCB3 protein, human; D054799:Phospholipase C beta; D020830:rac1 GTP-Binding Protein
keywords: Gβγ; PH domain; cell signaling; heterotrimeric G protein; phosphatidylinositol signaling; phospholipase C; protein conformation