Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors

Abstract

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Figure 1.

(a) Locations of cysteines in RGS4, RGS8, and RGS19. (b) Potency of CCG-50014 against RGS19, which has only one cysteine, and mutant RGS4 and RGS8 containing only the shared α4 helix cysteine. n=3.

Figure 2.

(a-e) Kinetics of deuterium exchange in selected protein fragments from (a) α4, (b) α5, (c) α5-α6 interhelical region, (d) α6 and (e) α7. Sequences of observed fragments are aligned and residue numbers of each fragment indicated. n=3.

Figure 3.

(a) Global kinetics of deuterium exchange. Deuterium incorporation is expressed as a percent of exchangeable amide hydrogen positions. (b) Degree of deuterium incorporation at 300 minutes in 90% D₂O is mapped onto protein structure of RGS4, RGS8, and RGS19. n=3.

Figure 4.

Root mean squared fluctuations (RMSF) per residue during 2-μs MD simulations of (a) RGS4 (PDB: 1AGR), (b) RGS8 (PDB: 2ODE), and (c) RGS19 (PDB: 1CMZ). The RMSF trends for each protein for the simulation set 2 are shown in Fig S2. Gray bars indicate helical regions.

Figure 5.

Solvent-accessible surface areas (SASA) are shown for sulfur atoms in shared cysteines on α4 helix for simulation set 1 (a) and set 2 (b) in RGS4, RGS8, and RGS19, and for shared cysteines on α6-α7 interhelical loop in simulation set 1 (c) and set 2 (d) in RGS4 and RGS8.

Figure 6.

Conformational changes during molecular dynamics simulations. Root mean square deviations of α6 helix and α6-α7 loop, starting conformation, and a snapshot conformation during MD simulation are shown for (a, d, g) RGS4, (b, e, h) RGS8, and (c, f, i) RGS19. Protein regions plotted in MD trajectories are depicted in color in protein structures. Arrows indicate locations of notable solvent exposure during simulation.