Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

Abstract

The transcriptional activator CprK1 from Desulfitobacteriumhafniense, 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.Halogenated hydrocarbons are often toxic molecules. They are widespread environmental pollutants because of their numerous applications, for example in industry (degreasers), agriculture (pesticides), and private households (flame retardants). Although these compounds are generally very stable, they can be converted in many sediments and soils by reductive dehalogenation. Several strictly anaerobic bacteria capable of dehalogenation have been isolated including Desulfomonile, Dehalobacter, and Desulfitobacterium. These organisms use chlorinated compounds as terminal electron acceptors (halorespiration) and thus remove the chloride atom while energy is conserved via electron transport phosphorylation (1-3). The prospect of using these organisms in bioremediation of sediments contaminated with a variety of chlorinated aromatic compounds is promising. Desulfitobacterium dehalogenans can reductively dechlorinate phenolic compounds at the ortho position (4, 5), and the closely related Desulfitobacterium hafniense DCB-2 (6) can dechlorinate phenolic compounds at the ortho and meta position; the latter reaction, however, is described only for 3,5-dichlorophenol (3,5-DCP) 2 as a substrate (7).A large number of the proteins involved in halorespiration are encoded in the chlorophenol reductive dehalogenase (cpr...

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…Most of rdh gene clusters are also associated with one rdhK subunit in various orientations. The rdhK-encoded proteins clearly belong to the large family of CRP/Fnr regulatory proteins from which CprK members of Desulfitobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were extensively studied and represent the paradigmatic DNA-binding regulatory protein for the respective chlorophenol reductive dehalogenase (cpr) operons [48][49][50][51][52][53][54][55][56] figure 3). First, a strong correlation could be established between the level of sequence identity (see also electronic supplementary material, table S7) and the genetic organization of the predicted rdh operons.…”

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

“…Rv2466c-To investigate the effect of the redox state on the conformation of Rv2466c, we first performed limited proteolysis experiments (36,37). As depicted in Fig.…”

Dual Roles of an Essential Cysteine Residue in Activity of a Redox-regulated Bacterial Transcriptional Activator

“…Based on studies of a related transcriptional activator (CprK1) from Desulfitobacterium hafniense, the optimal and consensus dehalobox sequence appears to be a perfect inverted repeat, TTAATX 4 ATTAA (11), that resembles the FNR consensus sequence (TTGATX 4 ATCAA) (12). The effector (a chlorinated aromatic) can bind to CprK in both the reduced and oxidized states, but the protein must be in the reduced state to bind DNA (13,14). Thus, dehalorespiration is regulated at the transcriptional level by effector binding and by redox state.…”

“…Based on the crystal structures of oxidized D. hafniense CprK1 and reduced D. dehalogenans CprK, an inactive state is stabilized by linkage of the effector domain to the DNAbinding domain by the intermolecular disulfide bond. In addition, mass spectrometric studies reveal that several regions of the structure exhibit enhanced dynamics upon reduction (14).…”

“…7, Cys 11 (in the oxidized protein) is very close to the recognition helix of the winged helix-turn-helix DNA-binding domain in CprK that binds to the dehalobox promoter sequence. Furthermore, binding of DNA protects the recognition helix (a 14-amino acid peptide including residues 182-196) from limited proteolysis (14). Although Cys 11 is not observed in the structure of the reduced protein, reduction of CprK increases the overall structural dynamics of CprK in the region of Cys 11 (14).…”