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. 2008 Oct;70(1):151-67.
doi: 10.1111/j.1365-2958.2008.06399.x. Epub 2008 Aug 20.

Molecular basis of halorespiration control by CprK, a CRP-FNR type transcriptional regulator

Colin Levy  1 , Katharine Pike, Derren J Heyes, M Gordon Joyce, Krisztina Gabor, Hauke Smidt, John van der Oost, David Leys
Affiliations

Molecular basis of halorespiration control by CprK, a CRP-FNR type transcriptional regulator

Colin Levy et al. Mol Microbiol. 2008 Oct.

Abstract

Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic compounds in a respiration-type metabolism. The transcriptional regulator CprK from Desulfitobacterium spp. induces expression of halorespiratory genes upon binding of o-chlorophenol ligands and is reversibly inactivated by oxygen through disulphide bond formation. We report crystal structures of D. hafniense CprK in the ligand-free (both oxidation states), ligand-bound (reduced) and DNA-bound states, making it the first member of the widespread CRP-FNR superfamily for which a complete structural description of both redox-dependent and allosteric molecular rearrangements is available. In conjunction with kinetic and thermodynamic ligand binding studies, we provide a model for the allosteric mechanisms underpinning transcriptional control. Amino acids that play a key role in this mechanism are not conserved in functionally distinct CRP-FNR members. This suggests that, despite significant structural homology, distinct allosteric mechanisms are used, enabling this protein family to control a very wide range of processes.

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Figures

Fig. 1
Fig. 1
Atomic structures of D. hafniense CprK in different states. A representative dimer is depicted for each structure in cartoon form. Individual structural elements are coloured as follows, the N-terminus (residues 1–18) in orange, the effector β-barrel domain (19–107) in blue, the central α-helices not affected in position by ligand binding (108–127) in red, the central α-helix region affected by ligand binding + loop region in purple (128–148), the DNA-binding domain (149–227) in teal, the C-terminus (228–232) in orange. In addition, the position of C11 and C200 is indicated (when visible) by yellow spheres. The bound OCPA ligand is depicted in atom coloured spheres with grey carbons. The bound DNA is depicted in atom spheres with nucleotides constituting the (de)halobox sequence in pale orange. In case of the oxidized CprK:OCPA complex a symmetry-related molecule is depicted in grey to illustrate the changed quaternary structure in this particular state. Dotted lines indicate the position of highly mobile linker elements or N/C termini not visible in the electron density maps. Structures are organized as follows, top left: CprKC200S; top right: oxidized CprK; middle left: CprKC200S:OCPA; middle right: oxidized CprK:OCPA (previously determined 2H6B; Joyce et al., 2006); bottom left: (de)halobox DNA: CprKC200S:OCPA complex.
Fig. 2
Fig. 2
Structure of the CprKC200S:OCPA complex. A. OCPA binding site. Key residues involved in OCPA binding are shown in atom coloured sticks (coloured coded with carbons coloured according to Fig. 1). The σA 2FoFc electron density map surround the bound OCPA molecule and is contoured at 1σ. In addition the Trp-106 side-chain is shown that is approximately ∼18 Å away from the bound OCPA. B. Overlay of distinct CprKC200S:OCPA dimer structures in two crystal forms (with and without glycerol). The ribbon traces are coloured according to Cα B-factor values. C. Overlay of CprKC200S:OCPA (in ribbon trace coloured according to Fig. 1) with the CRP:cAMP structure (in grey ribbon trace; PDB code 1G6N). The overlay is based on structural alignment of the respective central α-helices. D. Stereoview of the inter-domain contact established between a single DNA domain and the sensor binding domains. Key residues are depicted in atom coloured sticks, coloured coded as in Fig. 1.
Fig. 3
Fig. 3
Structure of the ligand-free CprKC200S. A. DNA domain interface. A stereo view of the DNA-binding domain interface with key residues represented in atom coloured sticks. B. Overlay of the CprKC200S ligand-free sensor domains (ribbon model coloured as in Fig. 1) with the corresponding CprKC200S:OCPA structure (in grey ribbons). Coloured arrows indicate relative motion of individual structural elements upon ligand binding. C. Stereoview of an overlay of the ligand binding sites and immediate environment for both CprKC200S and CprKC200S:OCPA structures. For clarity, labels in the left panel indicate the position of CprKC200S residues below the grey horizontal line and for CprKC200S:OCPA residues when above that line.
Fig. 4
Fig. 4
Dynamic information obtained from CprK crystal structures. A. Overlay of the different CprK ligand-free structures. The left panel displays an overlay of the D. hafniense CprKC200S and D. dehalogenans CprK (PDB code 2H6C) depicted in ribbons coloured coded according for Fig. 1. The right panel displays a similar overlay but for the oxidized D. hafniense CprK structure. B. Comparison between the CprKC200S ligand-free (to the left) and OCPA-soaked CprKC200S (to the right) structures. The ribbon models are coloured according to Cα B-factors.
Fig. 5
Fig. 5
Solution data for OCPA binding by D. hafniense CprKC200S. A. ITC analysis of CprKC200S OCPA interaction. The ITC experiment was carried out by titrating OCPA into the chamber containing CprK. This panel shows the raw heating power over time. B. The fit of the integrated energy values. C. Stopped-flow Trp fluorescence quenching. The top panel represents stopped-flow kinetic transients observed upon mixing various concentrations of OCPA with 1 μM CprK. An excitation wavelength of 295 nm was used and the fluorescence emission was measured using a 320 nm cut-off filter. Transients were recorded between 1 and 10 s at 0, 1, 5, 10, 25, 50, 100, 250 and 500 μM OCPA. The bottom panel depicts the OCPA concentration dependence of the observed rate. D. OCPA absorbance. Absorbance spectra of 5 μM OCPA were recorded in 25 mM Tris, 25 mM ethanolamine, 50 mM MES, 50 mM NaCl at pH 5.5 and pH 10.5. A significant red-shift in the absorbance maximum of the OCPA occurs upon deprotonation. The fluorescence emission spectrum of 1 μM CprK using an excitation wavelength of 295 nm is overlayed for a direct comparison.
Fig. 6
Fig. 6
Model for allosteric effects of OCPA binding by CprK. A schematic model illustrating the extreme positive cooperativity model for OCPA binding by CprK and associated structural reorganization. Individual structural elements are depicted in grey scale when observed to be highly mobile. Key amino acids and contacts are indicated where appropriate. For clarity, the DNA-binding domain hydrophobic set of residues involved in domain interactions (Leu-156, Leu-160, Leu-178, Met-176, Ile-186) is depicted as a black rectangle. The bound OCPA molecules are depicted in black (protonated) or red (deprotonated). We postulate binding of the first ligand is characterized by weak binding (Kd1∼2.5 mM) but following ligand deprotonation (as predicted by our pKa interrogation model; Joyce et al., 2006) and concomitant reorganization of the entire CprK a high affinity ligand binding site is created for the second ligand (Kd2∼1 μm). It is unclear whether deprotonation occurs prior to or during binding for the second ligand molecule (here depicted as binding in the deprotonated form).
Fig. 7
Fig. 7
(de)halobox DNA binding by CprKC200S:OCPA. A. An overlay of the CprKC200S:OCPA:DNA crystal structure with the available CRP:cAMP:DNA structure (PDB code 2CGP). The overlay is based on structural alignment of the respective HTH motives. CprK and associated DNA are depicted and colour coded as in Fig. 1. The CRP is similarly represented but in grey tones. B. Stereoview of DNA–CprK interface contact area. The DNA and key amino acids are represented in atom coloured sticks with carbons coloured according to Fig. 1. C. Schematic overview of the various CprKC200S DNA contacts established. The (de)halobox consensus sequence is depicted in bold while nucleotides that were not observed in the structure are depicted in grey scale. A black sphere indicates the base pairs for which geometric parameters deviate significantly from average. DNA backbone phosphates that make direct contact with CprK residues are depicted by a star.
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