Transcriptional activation by CprK1 is regulated by protein structural changes induced by effector binding and redox state.
The transcriptional activator CprK1 from Desulfitobacterium-hafniense, a member of the ubiquitous cAMP receptor protein/fumarate nitrate reduction regulatory protein family, activates transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. 3-chloro-4-hydroxyphenylacetate is a known effector for CprK1, which interacts tightly with the protein, and induces binding to a specific DNA sequence ("dehalobox," TTAAT--ATTAA) located in the promoter region of chlorophenol reductive dehalogenase genes. Despite the availability of recent x-ray structures of two CprK proteins in distinct states, the mechanism by which CprK1 activates transcription is poorly understood. In the present study, we have investigated the mechanism of CprK1 activation and its effector specificity. By using macromolecular native mass spectrometry and DNA binding assays, analogues of 3-chloro-4-hydroxyphenylacetate that have a halogenated group at the ortho position and a chloride or acetic acid group at the para position were found to be potent effectors for CprK1. By using limited proteolysis it was demonstrated that CprK1 requires a cascade of structural events to interact with dehalobox dsDNA. Upon reduction of the intermolecular disulfide bridge in oxidized CprK1, the protein becomes more dynamic, but this alone is not sufficient for DNA binding. Activation of CprK1 is a typical example of allosteric regulation; the binding of a potent effector molecule to reduced CprK1 induces local changes in the N-terminal effector binding domain, which subsequently may lead to changes in the hinge region and as such to structural changes in the DNA binding domain that are required for specific DNA binding.
Mazon
Hortense
H
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Gábor
Krisztina
K
Leys
David
D
Heck
Albert J R
AJ
van der Oost
John
J
van den Heuvel
Robert H H
RH
D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D045858:Desulfitobacterium / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D015533:Transcriptional Activation
D002373:Cyclic AMP Receptor Protein; D004247:DNA