Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins

Abstract

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.