Regulators of G-protein Signaling (RGS) proteins terminate G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating hydrolysis of GTP. Targeting RGS proteins with inhibitors is a strategy to increase receptor-mediated signaling. There are several existing RGS inhibitors, which including the thiadiazolidinones (TDZDs). All RGS inhibitors discovered to date are covalent modifiers of cysteine residues and these act preferentially on RGS4 over other RGS isoforms. To widen the scope of therapeutic potential of RGS inhibitors, it would be useful to have inhibitors with specificities for other isoforms. To aid in the development of new inhibitors, it will be important to understand what factors are responsible for RGS isoform selectivity. While RGS isoforms vary in their number and location of cysteines, cysteines that are shared among most RGS proteins are buried beneath the protein surface. We hypothesize that there is a dual role for cysteine complement and protein dynamics that drives specificity of TDZD inhibitors.Interestingly, representative RGS proteins RGS4, RGS8, and RGS19 have dramatic differences in potency of inhibition when mutated to contain a single cysteine. Hydrogen-deuterium exchange (HDX) was used to evaluate differences in flexibility among RGS proteins, and deuterium incorporation was found to be correlated with TDZD potency. Molecular dynamics studies supported these differences in flexibility, and illustrated that flexibility differences may underlie solvent accessibility of shared cysteines. To understand what structural elements control RGS domain flexibility, we focused on interhelical salt bridge-forming residues that differ among the RGS isoforms. Mutations that induced salt bridge formation in RGS19 decreased its flexibility and decreased potency of TDZD inhibition, while salt bridge removal in RGS8 and RGS4 increased flexibility and increased potency of inhibition. This suggests a causative relationship between protein dynamics and inhibitor potency. The movements observed in these proteins suggest that cysteines may be exposed to solvent by formation of a transient pocket, which may be taken advantage of in the design of non-covalent inhibitors. Finally, the role of individual conserved cysteines was evaluated. NMR studies of single-cysteine RGS8 mutants demonstrated that inhibitors can interact with either cysteine. Mass spectrometry studies showed that a TDZD inhibitor may mediate an interaction between the α4 and α7 cysteines in WT RGS8 by formation of a disulfide bond. As a whole, this work demonstrates a role for both cysteine interaction and protein dynamics in the control of RGS inhibitor selectivity.
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