Allosteric inhibition of the regulator of G protein signaling-Galpha protein-protein interaction by CCG-4986.
Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G protein-coupled receptor activation. RGS proteins exert their effect by directly binding to the activated Galpha subunit of the G protein, catalyzing the accelerated hydrolysis of GTP and returning the G protein to its inactive, heterotrimeric form. In previous studies, we have sought to inhibit this GTPase-accelerating protein activity of the RGS protein by using small molecules. In this study, we investigated the mechanism of CCG-4986 [methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitro-benzenesulfinimidoate], a previously reported small-molecule RGS inhibitor. Here, we find that CCG-4986 inhibits RGS4 function through the covalent modification of two spatially distinct cysteine residues on RGS4. We confirm that modification of Cys132, located near the RGS/Galpha interaction surface, modestly inhibits Galpha binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/Galpha interaction through an allosteric mechanism that almost completely inhibits the Galpha-RGS protein-protein interaction. These findings demonstrate three important points: 1) the modification of the Cys148 allosteric site results in significant changes to the RGS interaction surface with Galpha; 2) this identifies a "hot spot" on RGS4 for binding of small molecules and triggering an allosteric change that may be significantly more effective than targeting the actual protein-protein interaction surface; and 3) because of the modification of a positional equivalent of Cys148 in RGS8 by CCG-4986, lack of inhibition indicates that RGS proteins exhibit fundamental differences in their responses to small-molecule ligands.
Roman
David L
DL
Division of Medicinal and Natural Products Chemistry, University of Iowa College of Pharmacy, Iowa City, Iowa 52242, USA. david-roman@uiowa.edu
Blazer
Levi L
LL
Monroy
C Aaron
CA
Neubig
Richard R
RR
D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D011506:Proteins / Q000378:metabolism; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides
D020690:GTPase-Activating Proteins; D011506:Proteins; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled; D013449:Sulfonamides; C523871:methyl N-((4-chlorophenyl)sulfonyl)-4-nitrobenzenesulfinimidoate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D003545:Cysteine