PT  - JOURNAL ARTICLE
AU  - Vincent S. Shaw
AU  - Mohammadjavad Mohammadi
AU  - Josiah A. Quinn
AU  - Harish Vashisth
AU  - Richard R. Neubig
TI  - An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19
AID  - 10.1124/mol.119.117176
DP  - 2019 Dec 01
TA  - Molecular Pharmacology
PG  - 683--691
VI  - 96
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/96/6/683.short
4100  - http://molpharm.aspetjournals.org/content/96/6/683.full
SO  - Mol Pharmacol2019 Dec 01; 96
AB  - Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α4 helix of RGS4, 8, and 19 with salt bridge–modifying mutations. These results show that the α4 salt bridge–forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors.SIGNIFICANCE STATEMENT Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge–forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.