Structural basis for gene regulation by a B12-dependent photoreceptor

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.

Extended Data Figure 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 centered around 540 nm with a shoulder around 560 nm. 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 contoured around AdoCbl at 1.0 σ (gray). 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 shown in ribbons with helix bundle in yellow and Cbl-binding domain in green.

Extended Data Figure 2

The CarH DNA-binding domain is flexible in the absence of DNA. (a) Overlay of five CarH protomers, including the protomer shown in Figure 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 Figure 2a. DNA-binding domains are colored in dark cyan, light cyan, dark blue, black, and gray. 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 coloring as in (a).

Extended Data Figure 3

CarH mutant analysis and multiple sequence alignment. (a) Results summary for in vitro CarH mutant analysis. (b) Alignment of CarH sequences from different bacterial species. Sequence identity is shown in white font with red background, sequence similarity in red font. Colored triangles highlight functionally important positions, with filled triangles indicating residues analyzed by mutagenesis in this study and empty triangles indicating residues not analyzed 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 tetramerization. Coloring 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 dimer-dimer interface: cyan. Residues involved in more than one type of interaction are colored half/half. Residues at protein interfaces are less well conserved than other functionally important residues, likely 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 ESPript.

Extended Data Figure 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 A₂₈₀ (tracking protein) and A₅₂₂ (tracking Cbl) traces are shown. Molecular weights are calculated from the observed elution volumes as described in the 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 522 nm 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). Both of these mutant proteins can still bind Cbl (see 522 nm 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 (800 nM) as determined by EMSAs after incubation with AdoCbl (4 μM) in the dark. (h) EMSA data for WT CarH and the G160Q and G192Q mutants fit to the Hill equation, as described in the methods. K_D (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.

Extended Data Figure 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). 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 1-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 (800 nM) to DNA segments of different lengths after incubation with AdoCbl (4 μM) 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 (800 nM) and each of the 40-bp operator variants after incubation with AdoCbl (4 μM) 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 likely arise from oligomeric equilibria and residual amounts of light-exposed protein in the sample.

Extended Data Figure 6

CarH DNA binding, conformational changes upon binding, and comparison to BmrR. (a) 2F_o–F_c omit electron density for DNA-bound CarH, calculated after performing full refinement of the model with DNA omitted and contoured at 1.0 σ. 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 6 σ, 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 gray 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 neighboring DNA-binding domains, colored by domain. Each interface between two DNA-binding domains buries 280 Ų of surface from solvent on each DNA-binding domain. Interactions of Arg72 to Tyr7 and Glu11 are indicated by black dashed lines. Coloring 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, colored 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 modeled. 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 gray, remaining coloring as in (e). (i) BmrR bound to DNA (PDB ID code 1EXJ). 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.

Extended Data Figure 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 (tracking protein) and A₅₂₂ (tracking Cbl) traces are shown. Molecular weights are calculated from the observed elution volumes as described in the 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 (4 μM) in the dark.

Extended Data Figure 8

Light-exposed CarH has bis-His ligated Cbl. (a) Structure of light-exposed CarH including the DNA-binding domain (cyan) and other domains colored as in Figure 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 is shown in blue, contoured at 1.0 σ. (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 400 mM imidazole contains 60% bis-imidazole ligated Cbl and 40% Cbl with dimethylbenzimidazole and imidazole as ligands) 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 elsewhere. (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.

Extended Data Figure 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 time periods. For AdoCbl to bind to CarH and induce tetramerization, the photolyzed Cbl has to dissociate from the protein first. Thus, the extent of tetramer formation, as assessed by size exclusion chromatography (Superose 6 10/300 GL column), is indicative of the affinity of the protein for photolyzed Cbl. That is, lack of tetramer formation in the presence of fresh AdoCbl indicates that the photolyzed Cbl is still bound to the protein. The observed differential in tetramer formation between H132A and WT CarH is substantial: WT CarH retains its photolyzed Cbl, showing only a small amount of tetramer formation, whereas H132A CarH loses its photolyzed Cbl, reforming tetramers upon AdoCbl addition. (c) ESI-TOF mass spectra of WT and H132A CarH after light exposure also reveal differential affinity for photolyzed Cbl. Light-exposed WT CarH is 1329 Da larger in molecular weight than light-exposed H132A CarH, a number corresponding to the molecular weight of Cbl. This difference in molecular weight suggests that, even under the harsh conditions of this experiment, photolyzed 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 weights obtained (differing only due to 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.

Figure 1

Schematic of CarH-mediated light-dependent gene regulation. Structures of all three relevant states are reported herein. Red circles depict AdoCbl, red half moons photolyzed Cbl, and open red half moons 4′,5′- anhydroadenosine, the recently identified photolysis product of CarH-bound AdoCbl. See main text for details.

Figure 2

Structure of CarH protomer and comparison to MetH. (a) CarH protomer colored 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 (gray, PDB ID code 1BMT) and BmrR DNA-binding domain (red, PDB ID code 1EXJ). (c) MetH binds MeCbl (methyl group in yellow); modeling 5′-dAdo (cyan) results in steric clashes. (d) CarH accommodates AdoCbl through several substitutions compared to MetH. Cobalt-coordinating His in green. (e) Superposition of MetH and CarH, highlighting shift of the four-helix bundle between MetH (gray) and CarH (yellow). (f) Alignment of CarH and MetH sequences involved in binding the Cbl upper face, highlighting substitutions.

Figure 3

CarH oligomerization. (a) CarH protomers arranged in a head-to-tail dimer, colored by domain (helix bundle: yellow; Cbl-binding domain: green) with left protomer shown in lighter colors. AdoCbl shown as in Fig. 2a. DNA-binding domains hidden for clarity. (b) Close-up of selected residues at dimer interface. (c) Core of CarH tetramer, assembled from two head-to-tail dimers. Top dimer is colored as in (a) and bottom dimer is colored in black and gray. 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), colored as in (c). DNA-binding domains of colored dimer are shown in dark cyan, those of gray dimer are shown in light cyan. (f) Additional view of CarH tetramer, revealing how DNA-binding domains are positioned on the protein surface.

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 gray. 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 labeling (Extended Data Figure 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 due to the modest resolution of this structure, but many of the contacts shown are supported by mutagenesis (Extended Data Figure 7).

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 gray. DNA-binding domain is hidden for clarity (see Extended Data Figure 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. Colored 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. Coloring as in (a).