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Conformational dynamics of a regulator of G-protein signaling protein reveals a mechanism of allosteric inhibition by a small molecule.

ABSTRACT: Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the G?-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 that covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of apo RGS4 structures, and consistently find that the ?5-?6 helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-G? binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-G? complex in the presence of inhibitor suggest a relatively unstable protein-protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors.

SUBMITTER: Vashisth H

PROVIDER: S-EPMC3876963 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Conformational dynamics of a regulator of G-protein signaling protein reveals a mechanism of allosteric inhibition by a small molecule.

Vashisth Harish H Storaska Andrew J AJ Neubig Richard R RR Brooks Charles L CL

ACS chemical biology 20131024 12

Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 that covalently binds to cysteine residues ...[more]

PMID: 24093330

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Project description:HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.

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Conformational dynamics of a regulator of G-protein signaling protein reveals a mechanism of allosteric inhibition by a small molecule.

Publications

Conformational dynamics of a regulator of G-protein signaling protein reveals a mechanism of allosteric inhibition by a small molecule.

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