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Characterization of a TIR-like protein from Paracoccus denitrificans.

ABSTRACT: Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a alpha-helical coiled-coil. The NMR data indicates that the C-terminal TIR-like domain folds into a compact protein. Finally, using GST pull-down experiments, we show that the bacteria TIR-like domain binds to the mammalian receptor (TLR4) and adaptor (MyD88) TIR domains. We postulate that prokaryotic pathogens utilize the TIR-like proteins to interfere with the innate immune response of the mammalian host so that the bacterial infection can progress undetected.

SUBMITTER: Low LY

PROVIDER: S-EPMC1880877 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Characterization of a TIR-like protein from Paracoccus denitrificans.

Low Lieh Yoon LY Mukasa Takashi T Reed John C JC Pascual Jaime J

Biochemical and biophysical research communications 20070307 2

Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a ...[more]

PMID: 17362878

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Project description:Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+?N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

Dataset Information

Characterization of a TIR-like protein from Paracoccus denitrificans.

Publications

Characterization of a TIR-like protein from Paracoccus denitrificans.

Similar Datasets

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