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The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

ABSTRACT: FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the ?-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 ?, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.

SUBMITTER: Bonnet M

PROVIDER: S-EPMC3656280 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

Bonnet Mariette M Kurz Mareike M Mesa Socorro S Briand Christophe C Hennecke Hauke H Grütter Markus G MG

The Journal of biological chemistry 20130401 20

FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the α-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Her ...[more]

PMID: 23546876

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Project description:Symbiotic N(2) fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network. Part of it is a hierarchically arranged cascade in which the two-component regulatory system FixLJ, in response to a moderate decrease in oxygen concentration, activates the fixK(2) gene. The FixK(2) protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS) but also further regulatory genes (rpoN(1), nnrR, and fixK(1)). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, FixK(2), and FixK(1) regulons, which, respectively, consist of 26, 204, and 29 genes specifically regulated in microoxically grown cells. Most of these genes are subject to positive control. Particular attention was addressed to the FixK(2)-dependent genes, which included a bioinformatics search for putative FixK(2) binding sites on DNA (FixK(2) boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK(2) as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently oriented fixK(1) and cycS genes shared the same FixK(2) box for the activation of transcription in both directions. This recognition site may also be a direct target for the FixK(1) protein, because activation of the cycS promoter required an intact fixK(1) gene and either microoxic or anoxic, denitrifying conditions. We present evidence that cycS codes for a c-type cytochrome which is important, but not essential, for nitrate respiration. Two other, unexpected results emerged from this study: (i) specifically FixK(1) seemed to exert a negative control on genes that are normally activated by the N(2) fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a FixK(2)-dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosis-specific control.

Project description:BackgroundClassically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets.ResultsWe re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance.ConclusionsWe have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable structure that are suitable for follow-up structural studies. To our knowledge, we are the first to apply variable length motif models to eukaryotic ChIP-seq data sets and consequently the first to show their value in this domain. The results include a novel motif for the ubiquitous transcription factor Sp1.

Dataset Information

The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

Publications

The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

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