Structural basis for gene regulation by a B12-dependent photoreceptor

Journal name:
Nature
Volume:
526,
Pages:
536–541
Date published:
DOI:
doi:10.1038/nature14950
Received
Accepted
Published online

Abstract

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

At a glance

Figures

  1. Schematic of CarH-mediated light-dependent gene regulation.
    Figure 1: Schematic of CarH-mediated light-dependent gene regulation.

    Structures of all three relevant states are reported herein. Red circles depict AdoCbl, filled red semicircles photolysed Cbl, and open red semicircles 4′,5′- anhydroadenosine, the recently identified photolysis product of CarH-bound AdoCbl33. See main text for details.

  2. Structure of CarH protomer and comparison with MetH.
    Figure 2: Structure of CarH protomer and comparison with MetH.

    a, CarH protomer coloured by domain: N-terminal DNA-binding domain (cyan) with recognition helix (dark blue) and β-hairpin wing (purple) highlighted; central four-helix bundle (yellow); C-terminal Cbl-binding domain (green). AdoCbl shown with Cbl carbons in pink, 5′-dAdo group carbons in cyan, cobalt in purple. b, Overlay of CarH protomer with Cbl-binding module of MetH (grey, PDB accession number 1BMT17) and BmrR DNA-binding domain (red, PDB accession number 1EXJ38). c, MetH binds MeCbl (methyl group in yellow); modelling 5′-dAdo (cyan) results in steric clashes. d, CarH accommodates AdoCbl through several substitutions compared with MetH. Cobalt-coordinating His in green. e, Superposition of MetH and CarH, highlighting shift of the four-helix bundle between MetH (grey) and CarH (yellow). f, Alignment of CarH and MetH sequences involved in binding the Cbl upper face, highlighting substitutions.

  3. CarH oligomerization.
    Figure 3: CarH oligomerization.

    a, CarH protomers arranged in a head-to-tail dimer, coloured by domain (helix bundle: yellow; Cbl-binding domain: green) with left protomer shown in lighter colours. AdoCbl shown as in Fig. 2a. DNA-binding domains hidden for clarity. b, Close-up of selected residues at the dimer interface. c, Core of CarH tetramer, assembled from two head-to-tail dimers. Top dimer is coloured as in a; bottom dimer is coloured in black and grey. Gly160 and Gly192 at the dimer–dimer interface are shown as red spheres. Structure is from a sample of CarH that degraded during crystallization and lacks the DNA-binding domains (crystal form 2, see Methods). d, Close-up of Gly160 and Gly192 (red spheres) from two protomers at the dimer–dimer interface. e, Tetramer of full-length CarH including DNA-binding domains (crystal form 3), coloured as in c. DNA-binding domains of coloured dimer are shown in dark cyan, those of grey dimer are shown in light cyan. f, Additional view of CarH tetramer, revealing how DNA-binding domains are positioned on the protein surface.

  4. CarH DNA binding.
    Figure 4: CarH DNA binding.

    a, CarH tetramer bound to a 26-bp DNA segment (yellow). CarH is shown in ribbons with one head-to-tail dimer in green (Cbl-binding domains) and yellow (helix bundles) and the other dimer in grey. AdoCbl shown as in Fig. 2a. DNA-binding domains are shown in cyan. Sequence of DNA segment used for crystallization (larger font) as well as flanking sequences in the operator (smaller font) are shown below. Cyan bars indicate base pairs covered by each DNA-binding domain. Base pairs covered by the recognition helix are boxed; red box highlights the promoter −35 element. Nucleotides protected from hydroxyl radical cleavage are indicated by bullets. The orientation of the DNA in the structure was confirmed by heavy atom labelling (Extended Data Fig. 6b–d). b, Schematic of CarH–DNA interactions for the central DNA-binding domain, denoted as follows: black arrows, hydrogen bonds/electrostatic interactions; green arrows, van der Waals interactions; solid lines, contacts from protein side chains; dashed lines, contacts from main chain; (M), contacts in the DNA major groove; (m), contacts in the DNA minor groove. c, Close-up of interactions between CarH (cyan) and DNA (yellow). Hydrogen bonds and ionic interactions to the phosphate backbone are shown as black dashed lines. Contacts to DNA bases are shown in purple. Side-chain orientation is not unambiguous owing to the modest resolution of this structure, but many of the contacts shown are supported by mutagenesis (Extended Data Fig. 7).

  5. Light-induced conformational changes in CarH.
    Figure 5: Light-induced conformational changes in CarH.

    a, Structure of light-exposed CarH, with the helix bundle in orange and Cbl-binding domain in grey. DNA-binding domain is hidden for clarity (see Extended Data Fig. 8a). Cbl shown with carbons in pink and cobalt in purple. b, Structure of a CarH protomer in the dark state, shown with helix bundle in yellow, Cbl-binding domain in green, and 5′-dAdo group in cyan. Coloured lines highlight domain orientations. c, Light-induced helix bundle movement causes tetramer disassembly. Shown is a head-to-tail dimer of CarH in the dark state, but the right protomer is replaced by the structure of light-exposed CarH to show the steric clash. d, Departure of the 5′-dAdo group after light exposure leaves a large cavity on the Cbl upper face. The helix bundle (yellow) and the Cbl-binding domain (green) are shown in surface representation with selected residues shown as sticks. e, Helix bundle movement fills the cavity at the Cbl upper face and brings His132 to the cobalt, where it occupies the open coordination site. Colouring as in a.

  6. CarH crystals contain intact AdoCbl.
    Extended Data Fig. 1: CarH crystals contain intact AdoCbl.

    a, UV–vis spectra obtained from AdoCbl-bound CarH crystals at T=100 K (red trace) or AdoCbl-bound CarH in solution at T=298 K (black trace) exhibit good qualitative agreement and similar features, including a peak centred around 540nm with a shoulder around 560nm. Because many band intensities are orientation-dependent and the crystal spectrum changes with orientation but molecules are rotationally averaged in solution, quantitative comparison of the spectra is difficult. Note also that individual bands appear sharper in the crystal spectrum because the molecules have fewer rotational degrees of freedom and because fewer vibrational states are populated at T=100 K. b, Simulated annealing composite omit electron density (2.15 Å resolution) contoured around AdoCbl at 1.0σ (grey). The electron density covers the entire AdoCbl molecule including the Co–C bond, indicating that the Co–C bond remained intact during crystallization and data collection. AdoCbl is shown in stick representation with Cbl carbons in pink and 5′-dAdo group carbons in cyan. Co is shown as a purple sphere. The Co-coordinating His177 is shown in sticks with carbons in green. CarH is shown in ribbons with the helix bundle in yellow and Cbl-binding domain in green.

  7. The CarH DNA-binding domain is flexible in the absence of DNA.
    Extended Data Fig. 2: The CarH DNA-binding domain is flexible in the absence of DNA.

    a, Overlay of five CarH protomers, including the protomer shown in Fig. 2a, highlighting flexibility of DNA-binding domains. Structures are aligned by the Cbl-binding domains (green) and helix bundles (yellow) and shown in the same orientation as Fig. 2a. DNA-binding domains are coloured in dark cyan, light cyan, dark blue, black, and grey. AdoCbl is shown with Cbl carbons in pink, 5′-dAdo group carbons in cyan, and cobalt in purple. b–e, Individual CarH protomers shown side by side. Orientation and colouring as in a.

  8. CarH mutant analysis and multiple sequence alignment.
    Extended Data Fig. 3: CarH mutant analysis and multiple sequence alignment.

    a, Results summary for in vitro CarH mutant analysis. Table footnotes are as follows: *oligomerization was probed by SEC and DNA binding by gel shift analysis. Y30A, H42A: weakened binding at 100nM protein. G160Q, G192Q: dimer, no tetramer. §G160Q, G192Q: binds with reduced affinity and cooperativity and as a higher mobility (smaller size) complex. b, Alignment of CarH sequences from different bacterial species. Sequence identity is shown in white font with red background, sequence similarity in red font. Coloured triangles highlight functionally important positions, with filled triangles indicating residues analysed by mutagenesis in this study and empty triangles indicating residues not analysed by mutagenesis. Mutating the highly conserved His177 of the Cbl-binding motif, the lower axial ligand of bound AdoCbl, has previously been shown to impair AdoCbl binding and tetramerization8. Colouring is as follows: hydrogen bonds/ionic interactions to DNA, orange; contact to 5′-dAdo, green; histidines coordinating Cbl (His132 only coordinates after light exposure), red; hydrogen bonds/ionic interactions at dimer interface, black; hydrogen bonds/ionic interactions as well as Gly160 and Gly192 at the dimer–dimer interface, cyan. Residues involved in more than one type of interaction are coloured half/half. Residues at protein interfaces are less well conserved than other functionally important residues, probably because compensatory mutations and local structural deformations are possible. Note, however, that the T. thermophilus Arg176–Asp201 pair observed in our structure is inverted in Myxococcus xanthus, suggesting that the interaction is conserved. Alignment generated using ESPript64.

  9. Characterization of CarH mutants affecting oligomerization state.
    Extended Data Fig. 4: Characterization of CarH mutants affecting oligomerization state.

    a–f, SEC traces (Superdex 200 analytical SEC column) of CarH carrying mutations (a, b) near the 5′-dAdo group; c, d, at the head-to-tail dimer interface; and e, f, at the dimer–dimer interface. Shown are traces of mutants incubated with AdoCbl in the dark (top panels) and after light exposure (bottom panels). In all panels, both absorbance A280 nm (tracking protein) and A522 nm (tracking Cbl) traces are shown. Molecular masses are calculated from the observed elution volumes as described in Methods, and are consistent with a tetrameric species (137 kDa), a dimeric species (89 kDa), and a monomeric species (39 kDa). Notably, mutant CarH proteins that do not tetramerize in the presence of AdoCbl (D201R, H142A) also do not appear to bind AdoCbl (see 522nm traces of dark samples). This finding is consistent with previous studies that show cooperativity of AdoCbl binding and tetramerization, a feature that does not hold for other forms of Cbl (methylcobalamin, CNCbl and Cbl)8. Both of these mutant proteins can still bind Cbl (see 522nm traces of light-exposed samples), which further suggests that these mutants are properly folded and that the lack of AdoCbl binding stems from inability to oligomerize. Although the degree of tetramerization of CarH mutant proteins in the dark varied, all of these mutant proteins form Cbl-bound monomers after light exposure. g, DNA-binding capacity of WT and mutant proteins (800nM) as determined by EMSAs after incubation with AdoCbl (4µM) in the dark. h, EMSA data for WT CarH and the G160Q and G192Q mutants fit to the Hill equation, as described in Methods. Kd (in nM) and Hill coefficients from the fits are, respectively, (67±2) and (5.1±0.7) for WT CarH, (111±18) and (3.0±0.2) for G160Q CarH, and (253±17) and (2.5±0.3) for G192Q CarH. The data shown are the mean values and standard errors of three to five repeat experiments.

  10. Identification and validation of CarH operator sequence by EMSAs and footprinting.
    Extended Data Fig. 5: Identification and validation of CarH operator sequence by EMSAs and footprinting.

    a, Location of CarH operator in the intergenic region between carH and the carotenogenic crtB of the T. thermophilus genome. Structural and biochemical data are mapped onto the sequence. Three 11-bp CarH binding sites are shown in cyan font and the promoter −35 element is highlighted with a red box. Nucleotides protected from hydroxyl radical cleavage are indicated with bullets. The ~42-nucleotide DNase I footprint on the sense strand is shown above the sequence and that of the antisense strand has been omitted for clarity. Nucleotide numbering on the sense strand is relative to the carH transcription start site (underlined, +1)10. To identify suitable DNA constructs for crystallization, operator sequences were systematically trimmed around a ~40-bp segment, as indicated by the black bars, and binding was assessed by EMSAs (shown in b). The sequences of two 26-bp DNA segments used for co-crystallization are also shown. The blunt-ended 26-bp segment was used for determination of the CarH–DNA structure. The second 26-bp segment contained one-nucleotide 3′-overhangs and 5-iodo-deoxycytidine in position −25 (red) and was used to validate the mode of DNA binding. b, Binding of CarH (800nM) to DNA segments of different lengths after incubation with AdoCbl (4µM) in the dark. Substantial DNA binding was observed for a probe as small as 30-bp. c, DNase I and hydroxyl radical footprints of CarH on a 130-bp operator DNA segment. Disappearance of bands in the presence of CarH indicates protection from cleavage. Protected regions are marked on the side and were mapped onto the operator sequence using G + A chemical sequencing experiments performed in parallel. d, e, CarH binding to 40-bp operators carrying mutations. d, Sequences of tested operator variants. WT operator sequence shown at the top and bottom, with repeat sequences that CarH recognizes shown in cyan; 6-bp stretch contacted by CarH recognition helix is boxed. Mutations are as follows: Mut1–7: single (1–3), pairwise (4–6), and triple (7) mutations of AC to GT (positions 8/9); Mut8–14: single (8–10), double (11–13), and triple (14) mutations of (A/C)T to GC (positions 4/5); Mut15–18: pairwise (15–17) and triple (18) mutations of (A/G)A) to TT (positions 1/2). e, EMSAs with WT CarH (800nM) and each of the 40-bp operator variants after incubation with AdoCbl (4µM) in the dark. Note that two additional lower mobility complexes are observed, most apparent with the WT operator and its variants with comparable binding. The origin of these complexes is unknown, but they probably arise from oligomeric equilibria and residual amounts of light-exposed protein in the sample.

  11. CarH DNA binding, conformational changes upon binding, and comparison with BmrR.
    Extended Data Fig. 6: CarH DNA binding, conformational changes upon binding, and comparison with BmrR.

    a, 2FoFc omit electron density (3.89 Å resolution) for DNA-bound CarH, calculated after performing full refinement of the model with DNA omitted and contoured at 1.0σ. DNA is shown with carbons in yellow and recognition helix of a CarH DNA-binding domain with carbons in cyan. b, c, Validation of DNA-binding mode using heavy-atom-derivatized DNA segments. CarH was crystallized with a DNA segment containing 5-iodo-deoxycytidine in position −25 of the sense strand. Shown is the resulting anomalous difference density (purple mesh), contoured at 6σ, for both CarH–DNA complexes in the asymmetric unit, with peaks directly adjacent to the C5 atom of deoxycytidine in position −25. d, Chemical structure of 5-iodo-deoxycytidine. e, Comparison of CarH before and after DNA binding, revealing rearrangement of DNA-binding domains. CarH before DNA binding is shown with helix bundles and Cbl-binding domains in grey and DNA-binding domains in pink. CarH bound to DNA is shown with helix bundles and Cbl-binding domains in green and DNA-binding domains in cyan. The fourth DNA-binding domain of DNA-bound CarH is disordered and not modelled. DNA is shown in yellow. AdoCbl is shown with Cbl carbons in pink and 5′-dAdo group carbons in cyan. f, Contacts between residues in neighbouring DNA-binding domains, coloured by domain. Each interface between two DNA-binding domains buries 280Å2 of surface from solvent on each DNA-binding domain. Interactions of Arg72 to Tyr7 and Glu11 are indicated by black dashed lines. Colouring as in e. g, h, Models of individual CarH head-to-tail dimers bound to DNA. g, Head-to-tail dimer contributing the middle of the three DNA-binding domains, coloured by domain with DNA-binding domain in cyan, helix bundles in yellow, and Cbl-binding domains in green. The DNA-binding domain of the second protomer (right) is disordered and not modelled. DNA and AdoCbl are shown as in e. h, Head-to-tail dimer contributing the flanking DNA-binding domains. Helix bundles and Cbl-binding domains are shown in grey, remaining colouring as in e. i, BmrR bound to DNA (PDB accession number 1EXJ38). A BmrR dimer is shown in ribbon representation in orange and red. DNA is shown in yellow. BmrR binds as a dimer to a palindromic sequence and distorts the DNA double strand from its ideal conformation.

  12. In vitro characterization of CarH DNA binding mutants.
    Extended Data Fig. 7: In vitro characterization of CarH DNA binding mutants.

    a, b, SEC traces (Superdex 200 analytical SEC column) of CarH carrying mutations in the DNA-binding domain. Shown are traces of mutants (a) incubated with AdoCbl in the dark and (b) after light exposure. In all panels, both A280 nm (tracking protein) and A522 nm (tracking Cbl) traces are shown. Molecular masses are calculated from the observed elution volumes as described in Methods, and are consistent with a tetrameric species (137 kDa) and a monomeric species (39 kDa). c, DNA-binding capacity of mutants (800 nM) as determined by EMSAs after incubation with AdoCbl (4µM) in the dark.

  13. Light-exposed CarH has bis-His ligated Cbl.
    Extended Data Fig. 8: Light-exposed CarH has bis-His ligated Cbl.

    a, Structure of light-exposed CarH including the DNA-binding domain (cyan) and other domains coloured as in Fig. 5a. b, Close-up view of the Cbl in light-exposed CarH, with both coordinating His side chains shown in sticks. Simulated annealing composite omit electron density (2.65 Å resolution) is shown in blue, contoured at 1.0σ. (c) UV–vis spectra of light-exposed WT CarH (black) and H132A CarH (red) exhibit pronounced differences, indicating that the bis-His ligation is also formed in solution. d, UV–vis spectra of free OHCbl (50 µM) with increasing imidazole concentration. The spectrum of bis-imidazole ligated Cbl (black, Cbl with 400mM imidazole contains 60% bis-imidazole ligated Cbl and 40% Cbl with dimethylbenzimidazole and imidazole as ligands20) resembles that of light-exposed WT CarH, whereas the spectrum of free OHCbl (pink) resembles that of light-exposed H132A CarH. Note that the latter two are expected to be slightly different because free OHCbl contains a dimethylbenzimidazole group as the lower axial ligand, whereas light-exposed H132A CarH contains a histidine imidazole as the lower axial ligand. Experimental conditions chosen were similar to those reported elsewhere20. e, UV–vis spectra of AdoCbl-bound WT CarH (black) and H132A CarH (red) are virtually identical, suggesting that the mode of AdoCbl binding is unchanged, as is expected from the structure. f, g, Size-exclusion chromatograms (Superose 6 10/300 GL column) of AdoCbl-bound and light-exposed (f) WT CarH and (g) H132A CarH, demonstrating that H132A CarH, like WT CarH, forms a tetramer in the dark and undergoes light-dependent tetramer disassembly.

  14. Disruption of bis-His ligation by H132A mutation facilitates Cbl dissociation after photolysis.
    Extended Data Fig. 9: Disruption of bis-His ligation by H132A mutation facilitates Cbl dissociation after photolysis.

    a, b, WT and H132A CarH were exposed to light, rendering them monomeric, and then incubated with free AdoCbl at the indicated temperatures and periods. For AdoCbl to bind to CarH and induce tetramerization, the photolysed Cbl has to dissociate from the protein first. Thus, the extent of tetramer formation, as assessed by SEC (Superose 6 10/300 GL column), is indicative of the affinity of the protein for photolysed Cbl. That is, lack of tetramer formation in the presence of fresh AdoCbl indicates that the photolysed Cbl is still bound to the protein. The observed differential in tetramer formation between H132A and WT CarH is substantial: WT CarH retains its photolysed Cbl, showing only a small amount of tetramer formation, whereas H132A CarH loses its photolysed Cbl, reforming tetramers upon AdoCbl addition. c, ESI–TOF mass spectra of WT and H132A CarH after light exposure also reveal differential affinity for photolysed Cbl. Light-exposed WT CarH is 1,329 Da larger in molecular mass than light-exposed H132A CarH, a number corresponding to the molecular mass of Cbl. This difference in molecular mass suggests that, even under the harsh conditions of this experiment, photolysed Cbl remains bound to the WT CarH monomer but not to H132A CarH, indicating that Cbl dissociates more readily without the bis-His ligation. The minor peak next to WT CarH arises from protein bound to a potassium ion (mass shift 39 Da). Species marked with an asterisk correspond to an unidentified impurity in the H132A CarH sample. d, Control experiment showing ESI–TOF mass spectra of WT and H132A CarH in the AdoCbl-bound dark state. The very similar molecular masses obtained (differing only because of the H132A mutation) indicate that the mutation has no effect on AdoCbl binding, consistent with the fact that His132 is not coordinated to Cbl when the upper 5′-dAdo ligand is present. Both WT and H132A CarH lose their AdoCbl cofactor as the tetramer disassembles into monomeric units.

Tables

  1. Crystallographic data collection and refinement statistics
    Extended Data Table 1: Crystallographic data collection and refinement statistics

Accession codes

Primary accessions

References

  1. Ziegelhoffer, E. C. & Donohue, T. J. Bacterial responses to photo-oxidative stress. Nature Rev. Microbiol. 7, 856863 (2009)
  2. Eberhard, S., Finazzi, G. & Wollman, F. A. The dynamics of photosynthesis. Annu. Rev. Genet. 42, 463515 (2008)
  3. Kami, C., Lorrain, S., Hornitschek, P. & Fankhauser, C. Light-regulated plant growth and development. Plant Dev. 91, 2966 (2010)
  4. Palczewski, K. Chemistry and biology of vision. J. Biol. Chem. 287, 16121619 (2012)
  5. Zhang, E. E. & Kay, S. A. Clocks not winding down: unravelling circadian networks. Nature Rev. Mol. Cell Biol. 11, 764776 (2010)
  6. Purcell, E. B. & Crosson, S. Photoregulation in prokaryotes. Curr. Opin. Microbiol. 11, 168178 (2008)
  7. Möglich, A., Yang, X. J., Ayers, R. A. & Moffat, K. Structure and function of plant photoreceptors. Annu. Rev. Plant Biol. 61, 2147 (2010)
  8. Ortiz-Guerrero, J. M., Polanco, M. C., Murillo, F. J., Padmanabhan, S. & Elias-Arnanz, M. Light-dependent gene regulation by a coenzyme B12-based photoreceptor. Proc. Natl Acad. Sci. USA 108, 75657570 (2011)
  9. Perez-Marin, M. C., Padmanabhan, S., Polanco, M. C., Murillo, F. J. & Elias-Arnanz, M. Vitamin B12 partners the CarH repressor to downregulate a photoinducible promoter in Myxococcus xanthus. Mol. Microbiol. 67, 804819 (2008)
  10. Takano, H. et al. Involvement of CarA/LitR and CRP/FNR family transcriptional regulators in light-induced carotenoid production in Thermus thermophilus. J. Bacteriol. 193, 24512459 (2011)
  11. Elias-Arnanz, M., Padmanabhan, S. & Murillo, F. J. Light-dependent gene regulation in nonphototrophic bacteria. Curr. Opin. Microbiol. 14, 128135 (2011)
  12. Takano, H. et al. LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus. Microbiology 160, 26502660 (2014)
  13. Frey, P. A. in Comprehensive Natural Products II Chemistry and Biology Vol. 7 (eds Mander L. & Liu, H.-W.) 501546 (Elsevier, 2010)
  14. Banerjee, R. Radical carbon skeleton rearrangements: catalysis by coenzyme B12-dependent mutases. Chem. Rev. 103, 20832094 (2003)
  15. Brown, N. L., Stoyanov, J. V., Kidd, S. P. & Hobman, J. L. The MerR family of transcriptional regulators. FEMS Microbiol. Rev. 27, 145163 (2003)
  16. Navarro-Aviles, G. et al. Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor. Mol. Microbiol. 63, 980994 (2007)
  17. Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G. & Ludwig, M. L. How a protein binds B12: a 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266, 16691674 (1994)
  18. Diez, A. I. et al. Analytical ultracentrifugation studies of oligomerization and DNA-binding of TtCarH, a Thermus thermophilus coenzyme B12-based photosensory regulator. Eur. Biophys. J. 42, 463476 (2013)
  19. Cheng, Z., Li, K. R., Hammad, L. A., Karty, J. A. & Bauer, C. E. Vitamin B12 regulates photosystem gene expression via the CrtJ antirepressor AerR in Rhodobacter capsulatus. Mol. Microbiol. 91, 649664 (2014)
  20. Marques, H. M., Marsh, J. H., Mellor, J. R. & Munro, O. Q. The coordination of imidazole and its derivatives by aquocobalamin. Inorg. Chim. Acta 170, 259269 (1990)
  21. Linden, H. & Macino, G. White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa. EMBO J. 16, 98109 (1997)
  22. Froehlich, A. C., Liu, Y., Loros, J. J. & Dunlap, J. C. White collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 297, 815819 (2002)
  23. Nash, A. I. et al. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proc. Natl Acad. Sci. USA 108, 94499454 (2011)
  24. Burgie, E. S. & Vierstra, R. D. Phytochromes: an atomic perspective on photoactivation and signaling. Plant Cell 26, 45684583 (2014)
  25. Losi, A. & Gärtner, W. The evolution of flavin-binding photoreceptors: an ancient chromophore serving trendy blue-light sensors. Annu. Rev. Plant Biol. 63, 4972 (2012)
  26. Conrad, K. S., Manahan, C. C. & Crane, B. R. Photochemistry of flavoprotein light sensors. Nature Chem. Biol. 10, 801809 (2014)
  27. Herrou, J. & Crosson, S. Function, structure and mechanism of bacterial photosensory LOV proteins. Nature Rev. Microbiol. 9, 713723 (2011)
  28. Jenkins, G. I. The UV-B photoreceptor UVR8: from structure to physiology. Plant Cell 26, 2137 (2014)
  29. Nahvi, A., Barrick, J. E. & Breaker, R. R. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res. 32, 143150 (2004)
  30. Nahvi, A. et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 10431049 (2002)
  31. Johnson, J. E., Reyes, F. E., Polaski, J. T. & Batey, R. T. B. 12 cofactors directly stabilize an mRNA regulatory switch. Nature 492, 133137 (2012)
  32. Peselis, A. & Serganov, A. Structural insights into ligand binding and gene expression control by an adenosylcobalamin riboswitch. Nature Struct. Mol. Biol. 19, 11821184 (2012)
  33. Jost, M., Simpson, J. H. & Drennan, C. L. The transcription factor CarH safeguards use of adenosylcobalamin as a light sensor by altering the photolysis products. Biochemistry 54, 32313234 (2015)
  34. Girvan, H. M. & Munro, A. W. Heme sensor proteins. J. Biol. Chem. 288, 1319413203 (2013)
  35. Matthews, R. G. Cobalamin-dependent methyltransferases. Acc. Chem. Res. 34, 681689 (2001)
  36. Jarrett, J. T. et al. Protein radical cage slows photolysis of methylcobalamin in methionine synthase from Escherichia coli. Bioorg. Med. Chem. 4, 12371246 (1996)
  37. Bandarian, V., Ludwig, M. L. & Matthews, R. G. Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase. Proc. Natl Acad. Sci. USA 100, 81568163 (2003)
  38. Heldwein, E. E. Z. & Brennan, R. G. Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409, 378382 (2001)
  39. Watanabe, S., Kita, A., Kobayashi, K. & Miki, K. Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA. Proc. Natl Acad. Sci. USA 105, 41214126 (2008)
  40. Kutta, R. J. et al. The photochemical mechanism of a B12-dependent photoreceptor protein. Nature Commun. 6, 7907 (2015)
  41. Barker, H. A. et al. Isolation and Properties of crystalline cobamide coenzymes containing benzimidazole or 5,6-dimethylbenzimidazole. J. Biol. Chem. 235, 480488 (1960)
  42. Firth, R. A., Hill, H. A. O., Pratt, J. M., Williams, R. J. P. & Jackson, W. R. The circular dichroism and absorption spectra of some vitamin B12 derivatives. Biochemistry 6, 21782189 (1967)
  43. Hill, J. A., Williams, R. J. P. & Pratt, J. M. Chemistry of vitamin B12. Part I. The valency and spectrum of the coenzyme. J. Chem. Soc. 51495153 (1964)
  44. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307326 (1997)
  45. Kabsch, W. Xds. Acta Crystallogr. D 66, 125132 (2010)
  46. Sheldrick, G. M. & Schneider, T. R. Substructure solution with SHELXD. Acta Crystallogr. D 58, 17721779 (2002)
  47. Schneider, T. R. & Pape, T. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Cryst. 37, 843844 (2004)
  48. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215230 (2007)
  49. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 3042 (1996)
  50. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213221 (2010)
  51. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 21262132 (2004)
  52. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658674 (2007)
  53. Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439450 (2006)
  54. van Dijk, M. & Bonvin, A. M. J. J. 3D-DART: a DNA structure modelling server. Nucleic Acids Res. 37, W235W239 (2009)
  55. Ten Eyck, L. F. Fast Fourier transform calculation of electron density maps. Methods Enzymol. 115, 324337 (1985)
  56. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235242 (2011)
  57. Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 27282733 (2007)
  58. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 1221 (2010)
  59. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283291 (1993)
  60. Schrodinger, L. L. C. The PyMOL Molecular Graphics System v.1.3r1 (Schrodinger, LLC, 2010)
  61. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774797 (2007)
  62. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013)
  63. Jain, S. S. & Tullius, T. D. Footprinting protein-DNA complexes using the hydroxyl radical. Nature Protocols 3, 10921100 (2008)
  64. Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320W324 (2014)

Download references

Author information

  1. Present address: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, UK.

    • Juan Manuel Ortiz-Guerrero

Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA

    • Marco Jost,
    • Percival Yang-Ting Chen,
    • Gyunghoon Kang &
    • Catherine L. Drennan
  2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA

    • Catherine L. Drennan
  3. Howard Hughes Medical Institute, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA

    • Catherine L. Drennan
  4. Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain

    • Jésus Fernández-Zapata &
    • S. Padmanabhan
  5. Department of Genetics and Microbiology, Area of Genetics (Unidad Asociada al Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas), Faculty of Biology, Universidad de Murcia, Murcia 30100, Spain

    • María Carmen Polanco,
    • Juan Manuel Ortiz-Guerrero &
    • Montserrat Elías-Arnanz

Contributions

M.J. performed crystallographic experiments, J.F.-Z., M.C.P., and J.M.O.-G. performed in vitro mutant analyses, and G.K. and P.Y.-T.C. assisted with crystal structure refinement. M.J., S.P., M.E.-A., and C.L.D. designed experiments, analysed the data, and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank (PDB) under accession numbers 5C8A (AdoCbl-bound CarH crystal form 2), 5C8D (AdoCbl-bound CarH crystal form 3), 5C8E (AdoCbl- and DNA-bound CarH), and 5C8F (light-exposed CarH).

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: CarH crystals contain intact AdoCbl. (183 KB)

    a, UV–vis spectra obtained from AdoCbl-bound CarH crystals at T=100 K (red trace) or AdoCbl-bound CarH in solution at T=298 K (black trace) exhibit good qualitative agreement and similar features, including a peak centred around 540nm with a shoulder around 560nm. Because many band intensities are orientation-dependent and the crystal spectrum changes with orientation but molecules are rotationally averaged in solution, quantitative comparison of the spectra is difficult. Note also that individual bands appear sharper in the crystal spectrum because the molecules have fewer rotational degrees of freedom and because fewer vibrational states are populated at T=100 K. b, Simulated annealing composite omit electron density (2.15 Å resolution) contoured around AdoCbl at 1.0σ (grey). The electron density covers the entire AdoCbl molecule including the Co–C bond, indicating that the Co–C bond remained intact during crystallization and data collection. AdoCbl is shown in stick representation with Cbl carbons in pink and 5′-dAdo group carbons in cyan. Co is shown as a purple sphere. The Co-coordinating His177 is shown in sticks with carbons in green. CarH is shown in ribbons with the helix bundle in yellow and Cbl-binding domain in green.

  2. Extended Data Figure 2: The CarH DNA-binding domain is flexible in the absence of DNA. (268 KB)

    a, Overlay of five CarH protomers, including the protomer shown in Fig. 2a, highlighting flexibility of DNA-binding domains. Structures are aligned by the Cbl-binding domains (green) and helix bundles (yellow) and shown in the same orientation as Fig. 2a. DNA-binding domains are coloured in dark cyan, light cyan, dark blue, black, and grey. AdoCbl is shown with Cbl carbons in pink, 5′-dAdo group carbons in cyan, and cobalt in purple. b–e, Individual CarH protomers shown side by side. Orientation and colouring as in a.

  3. Extended Data Figure 3: CarH mutant analysis and multiple sequence alignment. (945 KB)

    a, Results summary for in vitro CarH mutant analysis. Table footnotes are as follows: *oligomerization was probed by SEC and DNA binding by gel shift analysis. Y30A, H42A: weakened binding at 100nM protein. G160Q, G192Q: dimer, no tetramer. §G160Q, G192Q: binds with reduced affinity and cooperativity and as a higher mobility (smaller size) complex. b, Alignment of CarH sequences from different bacterial species. Sequence identity is shown in white font with red background, sequence similarity in red font. Coloured triangles highlight functionally important positions, with filled triangles indicating residues analysed by mutagenesis in this study and empty triangles indicating residues not analysed by mutagenesis. Mutating the highly conserved His177 of the Cbl-binding motif, the lower axial ligand of bound AdoCbl, has previously been shown to impair AdoCbl binding and tetramerization8. Colouring is as follows: hydrogen bonds/ionic interactions to DNA, orange; contact to 5′-dAdo, green; histidines coordinating Cbl (His132 only coordinates after light exposure), red; hydrogen bonds/ionic interactions at dimer interface, black; hydrogen bonds/ionic interactions as well as Gly160 and Gly192 at the dimer–dimer interface, cyan. Residues involved in more than one type of interaction are coloured half/half. Residues at protein interfaces are less well conserved than other functionally important residues, probably because compensatory mutations and local structural deformations are possible. Note, however, that the T. thermophilus Arg176–Asp201 pair observed in our structure is inverted in Myxococcus xanthus, suggesting that the interaction is conserved. Alignment generated using ESPript64.

  4. Extended Data Figure 4: Characterization of CarH mutants affecting oligomerization state. (355 KB)

    a–f, SEC traces (Superdex 200 analytical SEC column) of CarH carrying mutations (a, b) near the 5′-dAdo group; c, d, at the head-to-tail dimer interface; and e, f, at the dimer–dimer interface. Shown are traces of mutants incubated with AdoCbl in the dark (top panels) and after light exposure (bottom panels). In all panels, both absorbance A280 nm (tracking protein) and A522 nm (tracking Cbl) traces are shown. Molecular masses are calculated from the observed elution volumes as described in Methods, and are consistent with a tetrameric species (137 kDa), a dimeric species (89 kDa), and a monomeric species (39 kDa). Notably, mutant CarH proteins that do not tetramerize in the presence of AdoCbl (D201R, H142A) also do not appear to bind AdoCbl (see 522nm traces of dark samples). This finding is consistent with previous studies that show cooperativity of AdoCbl binding and tetramerization, a feature that does not hold for other forms of Cbl (methylcobalamin, CNCbl and Cbl)8. Both of these mutant proteins can still bind Cbl (see 522nm traces of light-exposed samples), which further suggests that these mutants are properly folded and that the lack of AdoCbl binding stems from inability to oligomerize. Although the degree of tetramerization of CarH mutant proteins in the dark varied, all of these mutant proteins form Cbl-bound monomers after light exposure. g, DNA-binding capacity of WT and mutant proteins (800nM) as determined by EMSAs after incubation with AdoCbl (4µM) in the dark. h, EMSA data for WT CarH and the G160Q and G192Q mutants fit to the Hill equation, as described in Methods. Kd (in nM) and Hill coefficients from the fits are, respectively, (67±2) and (5.1±0.7) for WT CarH, (111±18) and (3.0±0.2) for G160Q CarH, and (253±17) and (2.5±0.3) for G192Q CarH. The data shown are the mean values and standard errors of three to five repeat experiments.

  5. Extended Data Figure 5: Identification and validation of CarH operator sequence by EMSAs and footprinting. (369 KB)

    a, Location of CarH operator in the intergenic region between carH and the carotenogenic crtB of the T. thermophilus genome. Structural and biochemical data are mapped onto the sequence. Three 11-bp CarH binding sites are shown in cyan font and the promoter −35 element is highlighted with a red box. Nucleotides protected from hydroxyl radical cleavage are indicated with bullets. The ~42-nucleotide DNase I footprint on the sense strand is shown above the sequence and that of the antisense strand has been omitted for clarity. Nucleotide numbering on the sense strand is relative to the carH transcription start site (underlined, +1)10. To identify suitable DNA constructs for crystallization, operator sequences were systematically trimmed around a ~40-bp segment, as indicated by the black bars, and binding was assessed by EMSAs (shown in b). The sequences of two 26-bp DNA segments used for co-crystallization are also shown. The blunt-ended 26-bp segment was used for determination of the CarH–DNA structure. The second 26-bp segment contained one-nucleotide 3′-overhangs and 5-iodo-deoxycytidine in position −25 (red) and was used to validate the mode of DNA binding. b, Binding of CarH (800nM) to DNA segments of different lengths after incubation with AdoCbl (4µM) in the dark. Substantial DNA binding was observed for a probe as small as 30-bp. c, DNase I and hydroxyl radical footprints of CarH on a 130-bp operator DNA segment. Disappearance of bands in the presence of CarH indicates protection from cleavage. Protected regions are marked on the side and were mapped onto the operator sequence using G + A chemical sequencing experiments performed in parallel. d, e, CarH binding to 40-bp operators carrying mutations. d, Sequences of tested operator variants. WT operator sequence shown at the top and bottom, with repeat sequences that CarH recognizes shown in cyan; 6-bp stretch contacted by CarH recognition helix is boxed. Mutations are as follows: Mut1–7: single (1–3), pairwise (4–6), and triple (7) mutations of AC to GT (positions 8/9); Mut8–14: single (8–10), double (11–13), and triple (14) mutations of (A/C)T to GC (positions 4/5); Mut15–18: pairwise (15–17) and triple (18) mutations of (A/G)A) to TT (positions 1/2). e, EMSAs with WT CarH (800nM) and each of the 40-bp operator variants after incubation with AdoCbl (4µM) in the dark. Note that two additional lower mobility complexes are observed, most apparent with the WT operator and its variants with comparable binding. The origin of these complexes is unknown, but they probably arise from oligomeric equilibria and residual amounts of light-exposed protein in the sample.

  6. Extended Data Figure 6: CarH DNA binding, conformational changes upon binding, and comparison with BmrR. (816 KB)

    a, 2FoFc omit electron density (3.89 Å resolution) for DNA-bound CarH, calculated after performing full refinement of the model with DNA omitted and contoured at 1.0σ. DNA is shown with carbons in yellow and recognition helix of a CarH DNA-binding domain with carbons in cyan. b, c, Validation of DNA-binding mode using heavy-atom-derivatized DNA segments. CarH was crystallized with a DNA segment containing 5-iodo-deoxycytidine in position −25 of the sense strand. Shown is the resulting anomalous difference density (purple mesh), contoured at 6σ, for both CarH–DNA complexes in the asymmetric unit, with peaks directly adjacent to the C5 atom of deoxycytidine in position −25. d, Chemical structure of 5-iodo-deoxycytidine. e, Comparison of CarH before and after DNA binding, revealing rearrangement of DNA-binding domains. CarH before DNA binding is shown with helix bundles and Cbl-binding domains in grey and DNA-binding domains in pink. CarH bound to DNA is shown with helix bundles and Cbl-binding domains in green and DNA-binding domains in cyan. The fourth DNA-binding domain of DNA-bound CarH is disordered and not modelled. DNA is shown in yellow. AdoCbl is shown with Cbl carbons in pink and 5′-dAdo group carbons in cyan. f, Contacts between residues in neighbouring DNA-binding domains, coloured by domain. Each interface between two DNA-binding domains buries 280Å2 of surface from solvent on each DNA-binding domain. Interactions of Arg72 to Tyr7 and Glu11 are indicated by black dashed lines. Colouring as in e. g, h, Models of individual CarH head-to-tail dimers bound to DNA. g, Head-to-tail dimer contributing the middle of the three DNA-binding domains, coloured by domain with DNA-binding domain in cyan, helix bundles in yellow, and Cbl-binding domains in green. The DNA-binding domain of the second protomer (right) is disordered and not modelled. DNA and AdoCbl are shown as in e. h, Head-to-tail dimer contributing the flanking DNA-binding domains. Helix bundles and Cbl-binding domains are shown in grey, remaining colouring as in e. i, BmrR bound to DNA (PDB accession number 1EXJ38). A BmrR dimer is shown in ribbon representation in orange and red. DNA is shown in yellow. BmrR binds as a dimer to a palindromic sequence and distorts the DNA double strand from its ideal conformation.

  7. Extended Data Figure 7: In vitro characterization of CarH DNA binding mutants. (149 KB)

    a, b, SEC traces (Superdex 200 analytical SEC column) of CarH carrying mutations in the DNA-binding domain. Shown are traces of mutants (a) incubated with AdoCbl in the dark and (b) after light exposure. In all panels, both A280 nm (tracking protein) and A522 nm (tracking Cbl) traces are shown. Molecular masses are calculated from the observed elution volumes as described in Methods, and are consistent with a tetrameric species (137 kDa) and a monomeric species (39 kDa). c, DNA-binding capacity of mutants (800 nM) as determined by EMSAs after incubation with AdoCbl (4µM) in the dark.

  8. Extended Data Figure 8: Light-exposed CarH has bis-His ligated Cbl. (331 KB)

    a, Structure of light-exposed CarH including the DNA-binding domain (cyan) and other domains coloured as in Fig. 5a. b, Close-up view of the Cbl in light-exposed CarH, with both coordinating His side chains shown in sticks. Simulated annealing composite omit electron density (2.65 Å resolution) is shown in blue, contoured at 1.0σ. (c) UV–vis spectra of light-exposed WT CarH (black) and H132A CarH (red) exhibit pronounced differences, indicating that the bis-His ligation is also formed in solution. d, UV–vis spectra of free OHCbl (50 µM) with increasing imidazole concentration. The spectrum of bis-imidazole ligated Cbl (black, Cbl with 400mM imidazole contains 60% bis-imidazole ligated Cbl and 40% Cbl with dimethylbenzimidazole and imidazole as ligands20) resembles that of light-exposed WT CarH, whereas the spectrum of free OHCbl (pink) resembles that of light-exposed H132A CarH. Note that the latter two are expected to be slightly different because free OHCbl contains a dimethylbenzimidazole group as the lower axial ligand, whereas light-exposed H132A CarH contains a histidine imidazole as the lower axial ligand. Experimental conditions chosen were similar to those reported elsewhere20. e, UV–vis spectra of AdoCbl-bound WT CarH (black) and H132A CarH (red) are virtually identical, suggesting that the mode of AdoCbl binding is unchanged, as is expected from the structure. f, g, Size-exclusion chromatograms (Superose 6 10/300 GL column) of AdoCbl-bound and light-exposed (f) WT CarH and (g) H132A CarH, demonstrating that H132A CarH, like WT CarH, forms a tetramer in the dark and undergoes light-dependent tetramer disassembly.

  9. Extended Data Figure 9: Disruption of bis-His ligation by H132A mutation facilitates Cbl dissociation after photolysis. (267 KB)

    a, b, WT and H132A CarH were exposed to light, rendering them monomeric, and then incubated with free AdoCbl at the indicated temperatures and periods. For AdoCbl to bind to CarH and induce tetramerization, the photolysed Cbl has to dissociate from the protein first. Thus, the extent of tetramer formation, as assessed by SEC (Superose 6 10/300 GL column), is indicative of the affinity of the protein for photolysed Cbl. That is, lack of tetramer formation in the presence of fresh AdoCbl indicates that the photolysed Cbl is still bound to the protein. The observed differential in tetramer formation between H132A and WT CarH is substantial: WT CarH retains its photolysed Cbl, showing only a small amount of tetramer formation, whereas H132A CarH loses its photolysed Cbl, reforming tetramers upon AdoCbl addition. c, ESI–TOF mass spectra of WT and H132A CarH after light exposure also reveal differential affinity for photolysed Cbl. Light-exposed WT CarH is 1,329 Da larger in molecular mass than light-exposed H132A CarH, a number corresponding to the molecular mass of Cbl. This difference in molecular mass suggests that, even under the harsh conditions of this experiment, photolysed Cbl remains bound to the WT CarH monomer but not to H132A CarH, indicating that Cbl dissociates more readily without the bis-His ligation. The minor peak next to WT CarH arises from protein bound to a potassium ion (mass shift 39 Da). Species marked with an asterisk correspond to an unidentified impurity in the H132A CarH sample. d, Control experiment showing ESI–TOF mass spectra of WT and H132A CarH in the AdoCbl-bound dark state. The very similar molecular masses obtained (differing only because of the H132A mutation) indicate that the mutation has no effect on AdoCbl binding, consistent with the fact that His132 is not coordinated to Cbl when the upper 5′-dAdo ligand is present. Both WT and H132A CarH lose their AdoCbl cofactor as the tetramer disassembles into monomeric units.

Extended Data Tables

  1. Extended Data Table 1: Crystallographic data collection and refinement statistics (269 KB)

Additional data