CprK Crystal Structures Reveal Mechanism for Transcriptional Control of Halorespiration

Abstract

Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the ligand orthochlorophenolacetic acid and of reduced CprK in absence of this ligand. These structures reveal that highly specific binding of chlorinated, rather than the corresponding non-chlorinated, phenolic compounds in the NH 2 -terminal ␤-barrels causes reorientation of these domains with respect to the central ␣-helix at the dimer interface. Unexpectedly, the COOH-terminal DNA-binding domains dimerize in the non-DNA binding state. We postulate the ligand-induced conformational change allows formation of interdomain contacts that disrupt the DNA domain dimer interface and leads to repositioning of the helixturn-helix motifs. These structures provide a structural framework for further studies on transcriptional control by CRP-FNR homologs in general and of halorespiration regulation by CprK in particular.Past and present industrial and agricultural activities have led to the ever increasing presence of haloorganic compounds such as chlorophenols and chlorinated ethenes in the environment (1). Due to both toxicity and recalcitrant nature, increasing amounts of these xenobiotics threaten the integrity of the environment and human health (2). In recent years, it has emerged that several haloorganic compounds are also naturally produced (3) and that several species of strictly anaerobic bacteria are able to conserve energy via the reductive dehalogenation of these compounds by respiratory metabolism (4, 5). In view of their favorable degrading capacities, e.g. high dehalogenation rate and low residual concentration of the contaminant, it has been anticipated that halorespiring microorganisms should be of utmost significance for efficient biological remediation of halogenated hydrocarbons in anoxic environments (6, 7). The versatile, strictly anaerobic Gram-positive bacterium Desulfitobacterium dehalogenans and the closely related Desulfitobacterium hafniense have the capacity of degrading ortho-chlorophenol. Both have been used as model organisms in halorespiration studies, representing one of the most significant groups of halorespiring isolates (8). In these organisms, proteins involved in halorespiration are encoded by the cpr (chlorophenol reductive dehalogenase) operon, of which multiple copies are present within the genome. This potentially allows for reductive dehalogenation of a wide range of haloorganic compounds by the use of a series of paralogous enzymes (9).The cpr operon is transcriptionally regulated by CprK, a member of the CRP-FNR family of regulators that is ubiquitous in bacteria (10). Recent in vivo and in vitro studies reveal that CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) 2 with micromolar affinity promoting a tight interaction with a specific DNA sequence in the promoter region of the cprencod...

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

“…In the x-ray structure of liganded oxidized CprK1 from D. hafniense, one CHPA molecule is bound per subunit in the ␤-barrel of the effector binding domain. The observed binding site is similar in position to the binding site of cAMP (3Ј,5Ј-cyclic adenosine monophosphate) in CRP (13). This structure is not compatible with tight binding to dehalobox dsDNA.…”

“…Comparison of the two crystal structures allowed us to postulate that the binding of CHPA to CprK1 causes reorientation of the N-terminal ␤-barrels with respect to the central ␣-helix at the dimer interface and with conformational changes in the C-terminal DNA binding domains as a consequence (13). The ultimate proof of this suggestion, i.e.…”

“…DNA binding studies have shown that CprK1 and CprK are only capable of binding its target DNA when the protein is in its reduced state (10,12). Recently, the x-ray structures of oxidized CprK1 from D. hafniense in complex with CHPA and reduced CprK from D. dehalogenans in its unliganded form have been determined by Joyce et al (13). They reveal that both CprKs exhibit high structural similarity to the cAMP receptor protein (CRP) (14,15) and PrfA, a key virulence regulator of Listeria monocytogenes (16).…”

“…Both CprKs bind CHPA with micromolar affinity promoting an interaction with dehalobox dsDNA (13). On the basis of the x-ray structures, it was suggested that: (i) the presence of the chloride group in the ortho position and (ii) the pK a interrogation mechanism (formation of phenolate) allow DNA binding.…”

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.…”