Role of the isoprenyl pocket of the G protein beta gamma subunit complex in the binding of phosducin and phosducin-like protein.
Phosducin (Pdc) and phosducin-like protein (PhLP) regulate G protein-mediated signaling by binding to the betagamma subunit complex of heterotrimeric G proteins (Gbetagamma) and removing the dimer from cell membranes. The binding of Pdc induces a conformational change in the beta-propeller structure of Gbetagamma, creating a pocket between blades 6 and 7. It has been proposed that the isoprenyl group of Gbetagamma inserts into this pocket, stabilizing the Pdc.Gbetagamma structure and decreasing the affinity of the complex for the lipid bilayer. To test this hypothesis, the binding of Pdc and PhLP to several Gbetagamma dimers containing variants of the beta or gamma subunit was measured. These variants included modifications of the isoprenyl group (gamma), residues involved in the conformational change (beta), and residues lining the proposed prenyl pocket (beta). Switching prenyl groups from farnesyl to geranylgeranyl or vice versa had little effect on binding. However, alanine substitution of one residue in the beta subunit involved in the conformational change (W332) decreased binding 5-fold. Alanine substitution of certain residues within the prenyl pocket caused only minor decreases in binding, while a lysine substitution of T329 within the pocket inhibited binding 10-fold. Molecular modeling of the binding energy of the Pdc.Gbeta(1)gamma(2) complex required insertion of the geranylgeranyl group into the prenyl pocket in order to accurately predict the effects of prenyl pocket amino acid substitutions. Finally, a dimer containing a gamma subunit with no prenyl group (gamma(2)-C68S) decreased binding by nearly 20-fold. These results support the structural model in which the prenyl group escapes contact with the aqueous milieu by inserting into the prenyl pocket and stabilizing the Pdc-binding conformation of Gbetagamma.
Lukov
Georgi L
GL
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
Myung
Chang-Seon
CS
McIntire
William E
WE
Shao
Jianyin
J
Zimmerman
S Scott
SS
Garrison
James C
JC
Willardson
Barry M
BM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005136:Eye Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000378:metabolism; D005822:Genetic Vectors; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017368:Protein Prenylation / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics
D002352:Carrier Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; C498320:Pdcl protein, rat; D010750:Phosphoproteins; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; C065631:phosducin