The Structure of Bradyrhizobium japonicum Transcription Factor FixK(2) Unveils Sites of DNA Binding and Oxidation
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2013-05
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Journal Article
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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 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 FixK ₂ 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 FixK ₂, 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 Å, 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. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
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288 (20)
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14238 - 14246
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American Society for Biochemistry and Molecular Biology
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03206 - Hennecke, Hauke (emeritus)