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Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis.

ABSTRACT: The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the active Rab1b state by locking the switch in the active signaling conformation independent of bound GTP or GDP and that electrostatic interactions due to the additional negative charge in the switch region make significant contributions. The stacking interaction between adenine and Phe45 however, seems to have only minor influence on this stabilisation. The results may also have implications for the mechanistic understanding of conformational switching in other signaling proteins.

SUBMITTER: Luitz MP

PROVIDER: S-EPMC4730224 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis.

Luitz Manuel P MP Bomblies Rainer R Ramcke Evelyn E Itzen Aymelt A Zacharias Martin M

Scientific reports 20160128

The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the activ ...[more]

PMID: 26818796

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Project description:BackgroundTaraxacum mongolicum (TM) is a widely used herb. Studies have reported that TM exhibits growth-inhibitory and apoptosis-inducing on multiple tumors, including hepatocellular carcinoma (HCC). The active ingredients, targets, and molecular mechanisms of TM against HCC need to be further elucidated.MethodsWe identified the active ingredients and targets of TM via HERB, PubChem, SwissADME, SwissTargetPrediction, and PharmMapper. We searched HCC targets from GeneCards, Comparative Toxicogenomics Database (CTD), and DisGeNET. Then, the intersection of drug targets and disease targets was uploaded to the STRING database to construct protein-protein interactions (PPI) networking whose topology parameters were analyzed in Cytoscape software to screen hub targets. Next, we used Metascape for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and we employed AutoDock vina, AMBER18 and PyMOL software along with several auxiliary tools for molecular docking and molecular dynamics (MD) simulation. Finally, based on the in silico findings, cellular experiments were conducted to investigate the effect of TM on HSP90AA1 gene expression.ResultsA total of 228 targets and 35 active ingredients were identified. Twenty two hub targets were selected through PPI networking construction for further investigation. The enrichment analysis showed that protein kinase binding, mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were mainly involved. Molecular docking and MD simulation results supported good interaction between HSP90 protein and Austricin/Quercetin. The in vitro assay showed that TM inhibited the proliferation of HepG2 cells and the expression of HSP90AA1 gene.ConclusionsThis study is the first to use network pharmacology, molecular docking, MD simulation and cellular experiments to elucidate the active ingredients, molecular targets, and key biological pathways responsible for TM anti-HCC, providing a theoretical basis for further research.

Project description:We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the C? sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 ?s (with one exception, where the trajectory length was 10 ?s) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.

Dataset Information

Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis.

Publications

Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis.

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