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An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

ABSTRACT: Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein ?-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 ? ₄ 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 ? ₄ 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 ? ₄ helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the ? ₄ 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.

SUBMITTER: Shaw VS

PROVIDER: S-EPMC6820219 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Shaw Vincent S VS Mohammadi Mohammadjavad M Quinn Josiah A JA Vashisth Harish H Neubig Richard R RR

Molecular pharmacology 20190922 6

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein <i>α</i>-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]- ...[more]

PMID: 31543506

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Project description:Halophilic proteins have greater abundance of acidic over basic and very low bulky hydrophobic residues. Classical electrostatic stabilization was suggested as the key determinant for halophilic adaptation of protein. However, contribution of specific electrostatic interactions (i.e. salt-bridges) to overall stability of halophilic proteins is yet to be understood. To understand this, we use Adaptive-Poison-Boltzmann-Solver Methods along with our home-built automation to workout net as well as associated component energy terms such as desolvation energy, bridge energy and background energy for 275 salt-bridges from 20 extremely halophilic proteins. We then perform extensive statistical analysis on general and energetic attributes on these salt-bridges. On average, 8 salt-bridges per 150 residues protein were observed which is almost twice than earlier report. Overall contributions of salt-bridges are -3.0 kcal mol-1. Majority (78%) of salt-bridges in our dataset are stable and conserved in nature. Although, average contributions of component energy terms are equal, their individual details vary greatly from one another indicating their sensitivity to local micro-environment. Notably, 35% of salt-bridges in our database are buried and stable. Greater desolvation penalty of these buried salt-bridges are counteracted by stable network salt-bridges apart from favorable equal contributions of bridge and background terms. Recruitment of extensive network salt-bridges (46%) with a net contribution of -5.0 kcal mol-1 per salt-bridge, seems to be a halophilic design wherein favorable average contribution of background term (-10 kcal mol-1) exceeds than that of bridge term (-7 kcal mol-1). Interiors of proteins from halophiles are seen to possess relatively higher abundance of charge and polar side chains than that of mesophiles which seems to be satisfied by cooperative network salt-bridges. Overall, our theoretical analyses provide insight into halophilic signature in its specific electrostatic interactions which we hope would help in protein engineering and bioinformatics studies.

Dataset Information

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Publications

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

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