Investigating Regulator of G‐protein Signaling (RGS) Protein Dynamics by Hydrogen/Deuterium Exchange
Authors: Shaw Vincent S. , Schilmiller Anthony L. , Vashisth Harish , Neubig Richard R. 
Published: June 2021

Regulator of G‐protein Signaling (RGS) proteins play a key regulatory role in G‐protein coupled receptor (GPCR) signaling by accelerating hydrolysis of GTP bound to an active G protein's alpha subunit, thus terminating signaling. There are over 20 different members of the RGS protein family, each expressed at different levels in different tissues. By pharmacologically targeting RGS proteins, G protein signaling may be modulated with greater tissue specificity than by application of a GPCR agonist alone. In particular, RGS4 may be a useful therapeutic target for treatment of Parkinson's disease. Thiadiazolidinones, a series of compounds that inhibit RGS4, have been shown to reduce bradykinesia in a raclopride‐induced mouse model of Parkinson's disease (Blazer et al., 2015). Thiadiazolidinones have been found to be most potent against RGS4, followed by RGS19 and RGS8, and inhibit by covalent modification of cysteine residues. However, cysteines shared by these isoforms are not exposed to the protein surface. Our hypothesis is that variation in flexibility between RGS isoforms causes differential transient exposure of otherwise buried cysteines, affecting thiadiazolidinone selectivity. Gaining a better understanding of the mechanism of thiadiazolidinone binding could inform future rational design of RGS inhibitors.Hydrogen/Deuterium Exchange, or HDX, is a tool for gauging protein dynamics that takes advantage of spontaneous exchange between solvent deuterium and amide hydrogens of the protein backbone. Deuterium incorporation over time was measured in purified His‐tagged RGS domains of RGS4, RGS8, and RGS19. After 1000 min, deuterium incorporation in helix 4, which contains the only cysteine shared by all three RGS isoforms, had reached 35% in RGS4, but only 9% RGS8. Across helices 4, 5, and 6, RGS19 had much faster deuterium incorporation than RGS4 or RGS8. For example, in a fragment of helix 5, RGS19 had 51% deuterium incorporation at 30 min, while similarly located fragments in RGS4 and RGS8 had 15% and 17% incorporation, respectively. Thermal Stability was also assessed. RGS4 was found to be the least thermally stable, with a melting temperature of 50°C, followed by RGS19 and RGS8, with melting temperatures of 53°C and 54°C respectively. The low thermal stability of RGS4 suggests increased flexibility, providing an explanation for the selectivity of thiadiazolidinones for RGS4 over other RGS isoforms. Greater helix 4 deuterium incorporation in RGS4 than in RGS8 also suggests increased solvent exposure, leading to better access by thiadiazolidinone inhibitors. However, RGS19, despite having the most extensive deuterium incorporation, is not the most potently inhibited. This may be because RGS19 lacks cysteines that play strong roles in inhibition of RGS4. These results indicate that protein flexibility is not the sole determinant of potency of RGS protein inhibition by thiadiazolidinones.Support or Funding InformationThis work is supported by NSF grant 1507588 and NIH T32 grant GM092715.