Photocobilins integrate B12 and bilin photochemistry for enzyme control

Zhang, Shaowei; Jeffreys, Laura N.; Poddar, Harshwardhan; Yu, Yuqi; Liu, Chuanyang; Patel, Kaylee; Johannissen, Linus O.; Zhu, Lingyun; Cliff, Matthew J.; Yan, Cunyu; Schirò, Giorgio; Weik, Martin; Sakuma, Michiyo; Levy, Colin W.; Leys, David; Heyes, Derren J.; Scrutton, Nigel S.

doi:10.1038/s41467-024-46995-1

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Published: 28 March 2024

Photocobilins integrate B₁₂ and bilin photochemistry for enzyme control

Nature Communications volume 15, Article number: 2740 (2024) Cite this article

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Abstract

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B₁₂) can act as a light-sensing chromophore heralded a new field of B₁₂-photobiology. Although microbial genome analysis indicates that photoactive B₁₂-binding domains form part of more complex protein architectures, regulating a range of molecular–cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B₁₂ and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B₁₂ and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B₁₂ affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B₁₂ photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.

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Introduction

Photoreceptor proteins are ubiquitous in nature and regulate a large range of biological processes in response to light. They have become essential components for optogenetic applications, where they can be fused to different output domains to provide light control of various cellular functions¹. The number of known photoreceptor families in biology has recently been expanded² by the discovery of a new superfamily of B₁₂ photoreceptors that repurpose coenzyme B₁₂ or adenosylcobalamin (AdoCbl) for light sensing. Cobalamins are complex cobalt-containing tetrapyrrole molecules, in which various forms differ in the nature of the upper axial ligand (e.g., methyl, cyano, hydroxyl or adenosyl) that is ligated to the central Co atom. For a long time, cobalamin has only been known as a widespread organometallic cofactor to many thermally-driven enzymes that catalyse a wide range of processes essential to living organisms, including humans³. The discovery of CarH, the canonical AdoCbl photoreceptor, revealed B₁₂ can support a light-mediated repression of carotenoid biosynthetic genes⁴. In the dark, the AdoCbl-bound CarH tetramer specifically binds to DNA and blocks transcription. On exposure to light, the photolabile Co-C bond of AdoCbl is cleaved, which results in the release of free 4’,5’-anhydro-adenosine^4,5. B₁₂ photochemistry triggers structural changes culminating in the formation of a bis-His ligated light state of CarH and the dissociation of the tetramer, together with the concomitant release from DNA and ultimately transcription initiation⁴. CarH has already been used in several green light-dependent biotechnological applications, such as in the formation of light-responsive hydrogels for drug delivery^6,7,8,9, light-activated technology devices¹⁰ and the regulation of mammalian gene expression¹¹. However, the optogenetic potential of B₁₂ photoreceptors would be significantly enhanced by broadening the wavelength range to include the more penetrative red/far-red region of the spectrum. More complex putative B₁₂ photoreceptors have since been identified in genomes, but lack functional characterisation^12,13. Sequence analysis reveals many CarH-like homologues form considerably more complex protein architectures, with the B₁₂-binding domain fused to additional enzyme or chromophore-binding domains¹². Based on these previous findings, we describe a new subfamily of B₁₂-dependent photoreceptors, consisting of a B₁₂ binding domain fused to a globin-like (or photoglobin⁶) domain. The hypothesis that these coupled photoglobin and B₁₂-binding domains may act as a light-sensing regulatory bundle¹³ provides exciting opportunities to develop new optogenetic tools. We now demonstrate that these proteins can simultaneously bind B₁₂ and the linear tetrapyrrole biliverdin (BV), and are sensitive to light across the entire visible spectrum (Fig. 1). Hence, we propose these form a new family of photocobilins (photoactive-cobalamin-bilin, Pcob) that can mediate light-regulation of a range of processes.

**Fig. 1: The chromophores and wavelength sensitivity of the new multi-centre photocobilin photoreceptors.**

Results and discussion

Photocobilin proteins exhibit spectral changes upon light activation

We have identified and characterised two types of photocobilins, one from Saccharothrix syringae (SasPcob) that represents a standalone photocobilin photoreceptor and a more complex protein that consists of a photocobilin diguanylate cyclase (DGC) fusion from Acidimicrobiaceae bacterium (AbDPcob) that is representative of a photocobilin domain fused to an enzyme domain (Fig. 1 and Supplementary Figs. 1 and 2). Both proteins are able to bind AdoCbl and BV, although there are significant differences in BV binding between the two proteins (Figs. 2a, b, 3). In SasPcob, the ternary AdoCbl-BV complex is readily formed when both chromophores are provided. AdoCbl binding allosterically affects BV affinity, with the BV dissociation constant decreasing ~10-fold from K_d ~ 7 µM to ~0.7 µM in the presence of AdoCbl (Fig. 3 and Supplementary Fig. 3). In the full-length AbDPcob, full occupancy of AdoCbl is observed in the absence of BV, but the binding of both chromophores simultaneously is diminished compared to the SasPcob (Fig. 3 and Supplementary Fig. 3). The weaker binding and lower occupancy levels (~20%) of BV in AbDPcob suggest that evolutionary pressures may have reduced the requirement for BV and resulted in a different role for the globin-like domain in these more complex fusion proteins. Despite these differences, both the standalone photocobilin (SasPcob) and diguanylate cyclase fusion (AbDPcob) show similar light-sensing behaviour (Fig. 2c, d and Supplementary Fig. 4a, b). Illumination of AdoCbl-only bound photocobilin with red light (660 nm LED) has no effect (Fig. 2 and Supplementary Fig. 4), whereas illumination with green light (530 nm LED) elicits spectral changes similar to those observed previously with CarH¹⁴. These include the formation of new absorbance features at 356 nm and 500 nm, together with the disappearance of the absorbance band at approximately 570 nm. In contrast, the BV-only bound species does not display any obvious spectral changes after illumination with green or red light (Fig. 4a, b). However, the ternary AdoCbl-BV-photocobilin complexes respond to both green and red light, with excitation of the BV chromophore leading to spectral changes associated with Co-C bond cleavage in the B₁₂ cofactor (Fig. 2 and Supplementary Fig. 4). Hence, the two chromophores are optically coupled and excitation of either can trigger AdoCbl photoconversion, thereby expanding light sensitivity to cover most of the visible spectrum. The relative efficiency of conversion to the light state is higher for the direct AdoCbl excitation route (Supplementary Fig. 5). The ability to respond to red light is lost when AdoCbl is replaced with methylcobalamin, which contains a smaller methyl group as upper axial ligand (Fig. 4c−h). This indicates that the upper adenosyl ligand is required for optical coupling and cleavage of the upper axial C-Co bond. However, the exact mechanism of coupling between the 2 chromophores is currently not clear and will require further characterisation.

**Fig. 2: The light response and structure of the photocobilin photoreceptors.**

**Fig. 3: Chromophore binding properties of photocobilins.**

**Fig. 4: Light response of photocobilin proteins with methycobalamin bound.**

X-ray crystallography provides a structural rationale for photoactivation in photocobilins

To provide a structural rationale for the photocobilin photo-sensing properties, we determined the crystal structures of the dark (PDB 8J2W) and light-adapted states (PDB 8J2X) of SasPcob, as well as the dark state of the isolated AbDPcob photoreceptor domain (AbPcob, PDB 8J2Y), at 1.7 Å;, 2.0 Å; and 2.3 Å;, respectively (Figs. 2e, f, 5a−d, Table 1 and Supplementary Fig. 6). Unfortunately, we were unable to obtain crystals of the full length AbDPcob protein or the light state of AbPcob. Despite the low sequence identity (~34%), the structures of the two photocobilin proteins are similar, with an RMSD value of 1.63 Å for 464 C-alphas after structural alignment. Both the SasPcob and AbPcob proteins contain the W-(10)x-EH and E/DxH motifs (Fig. 5e) in the B₁₂-binding domain, which is found in other light dependent CarH-like B₁₂ photoreceptors¹⁵. The arrangement and dimerisation of the B₁₂ binding domains, which consist of a Rossmann fold and associated four-helix bundle, is similar to the corresponding CarH dimer core module (RMSD = 1.37 for 237 C-alphas), with the dark state SasPcob and AbPcob forming head-to-tail dimers⁴ (Fig. 5d, e). AdoCbl binding is also reminiscent of CarH, with the dimethylbenzimidazole tail embedded in the Rossmann fold (Figs. 2e, f, 6a), while the conserved His108 (SasPcob numbering) provides the lower axial ligand to the Co atom of the B₁₂ cofactor (i.e., base-off His-on ligation, Fig. 7a−c). The adenosine binding pocket is strikingly similar, with the ribose moiety stacking with the conserved Trp61 residue and forming a network of polar interactions linking across the dimer interface via the conserved Glu71-His72 motif (Figs. 2e, f, 6a). The Pcob specific C-terminal five-helix globin domain does not contribute directly to the dimer interface, and is positioned such that it forms interactions with both of the B₁₂-binding domains (i.e., Rossmann fold and four-helix bundle). This arrangement effectively shields much of the corrin ring edge that is normally solvent exposed when bound by the canonical CarH domain.

**Fig. 5: Structural comparison of photocobilins with CarH⁴ and BV binding protein^20,21.**

Table 1 Data collection and refinement statistics (molecular replacement) for Pcobs

Full size table

**Fig. 6: Chromophore binding of *Sas*Pcob and BV docking into AbPcob.**

**Fig. 7: Comparison of AdoCbl binding and light induced changes in photocobilin and CarH proteins.**

In SasPcob, the BV is bound with a ZZZ configuration, with Asp270 forming key interactions with the co-planar A, B and C pyrrole rings (Fig. 6a, b). In comparison to other BV-binding photoreceptors, notably the bacteriophytochromes, the photocobilins lack the Cys residue that covalently links to the BV molecule and is essential for light-induced cis-trans isomerisation^16,17,18 (Figs. 2e, f and 5f−i). Despite the lack of cysteinyl-linkage, the structure of the SasPcob BV-binding pocket is akin to light-harvesting phycobilisome proteins, with the BV orientation flipped by ~180° (Fig. 5g), projecting the D ring out of the globin core, as opposed to the A ring in other bilin binding proteins^19,20,21. The D ring is positioned out of the ABC plane by approximately 40° due to close interactions with Trp296 (Figs. 5g and 6a, b). Although the putative BV binding globin scaffold is retained in AbPcob, the reduced BV affinity is most likely due to replacement of a key BV binding residue, Asp270 in SasPcob, with a His (Fig. 5f−i). The crystal structure of AbPcob revealed that steric hindrance by this His residue interferes with BV binding, unlike in SasPcob. Molecular dynamics simulations and ligand docking (Supplementary Figs. 7–9, Supplementary Table 1 and 2, and Supplementary Data 1) demonstrate that rearrangement of surrounding residues could allow BV to bind with AbPcob in certain conformations, where hydrophobic interactions with residues in the binding pocket (e.g., Leu252, Trp289, Leu294, Arg297, Val299 and Val303) contribute to retain BV in the correct orientation (Fig. 6c−h). The lack of the main binding interaction with the Asp residue in AbPcob may explain the absence of any obvious spectral shift and increase in absorbance at 700 nm upon BV binding that is observed for SasPcob (Fig. 2). These findings could explain the lower BV occupancy levels for AbPcob whilst retaining the ability to respond to red light (Fig. 3 and Supplementary Fig. 4).

In SasPcob, the close interaction and relative position of the B₁₂- and BV-binding domains brings the two chromophores in close proximity, with the edge of the BV C and D rings within van der Waal’s distance of the conserved Trp61 and the corrin ring B, respectively. Furthermore, a direct hydrogen bond is formed between one of the corrin ring B amide groups and the BV ring D (Fig. 2e, f). This likely explains the B₁₂ allosteric effects on BV affinity observed in solution. A network of direct and water mediated polar interactions surround this central chromophore edge-to-edge contact. These include direct hydrogen bonds between the corrin ring and Trp296 from the globin domain, and a putative salt bridge formed between the propionic acid of ring C of BV and His137 of the opposite monomer (Fig. 2e). The juxtaposition of both chromophores likely underpins the optical coupling observed.

In order to investigate the effects of illumination on the photocobilins, we determined the light-exposed SasPcob crystal structure (Figs. 5, 7). Similar to CarH, the SasPcob light state corresponds to a monomeric species (Supplementary Fig. 1), with the B₁₂-binding domain dimer interface disrupted by the light-triggered release of the adenosine group (Fig. 7 and Supplementary Figs. 10−21). However, the SasPcob light state structure lacks the drastic large-scale domain repositioning that accompanies formation of a bis-His ligated cobalamin in CarH. Instead, more modest changes occur at the adenosine binding pocket in SasPcob culminating with the formation of a water/hydroxide ligated Co(III)balamin. While the majority of the Rossmann and globin binding domains are relatively unperturbed, the N-terminal four helix bundle adapts to the removal of the adenosine through changes in the position of the Glu71/His72 containing helix. The C-alpha displacement of ~2.4 Å at the Glu71/His72 region is such that, when superposed on the corresponding dark SasPcob dimer, it would lead to a severe clash at the dimer interface (Fig. 7c). Therefore, we postulate that rearrangement of the adenosine binding pocket in response to illumination occurs concomitantly with monomer formation.

Light-induced functional changes in photocobilins

To investigate how the photocobilin light-response can affect functional change, we investigated the activity of the AbDPcob DGC domain. This domain catalyses the formation of cyclic-di-GMP, involved in regulating a number of cellular processes^22,23,24, from GTP with the release of pyrophosphate. We successfully expressed the full-length AbDPcob protein and measured DGC activity through the detection of both cyclic-di-GMP and pyrophosphate (Fig. 8 and Supplementary Tables 4 and 5). Initially, the production of cyclic-di-GMP, confirmed by LC-MS, was found to increase upon illumination when compared to the apo (i.e., no chromophore bound) and AdoCbl-bound dark states of the protein (Fig. 8a). Steady-state kinetics were measured using a pyrophosphate assay for the apo and AdoCbl-bound dark/light states of the AbDPcob protein, as well as the truncated DGC domain only (Fig. 8b). Although the Michaelis constant, K_m, for GTP is similar for all protein forms, the apparent turnover number, k_cat, varies significantly and suggests major differences in DGC activity (Fig. 8b). All full-length AbDPcob forms are more active than the isolated DGC domain, indicating that the presence of the photocobilin domain plays a role in maintaining the DGC activity. The binding of AdoCbl to the full-length protein results in a decrease in DGC activity compared to the apo protein. However, upon illumination, there is a > 10-fold increase in apparent turnover number, implying that photoconversion of the AdoCbl-bound photocobilin domain to the light state activates the enzymatic activity of the neighbouring DGC domain. Simple replacement of the AdoCbl with the smaller upper ligand found in methylcobalamin, hydroxycobalamin or cyanocobalamin does not increase DGC activity (Fig. 8c). This implies that the presence of the adenosyl group or changes mediated through AdoCbl photochemistry that lead to the removal of the adenosyl group are important in this dark-inhibition and light-activation process. The oligomerisation state of the protein is not affected by GTP binding so this process is light-mediated (Supplementary Fig. 22).

**Fig. 8: The photoactivation and regulation of enzyme activity by the photocobilins.**

Our attempts to crystallise the full-length AbDPcob protein were unsuccessful, most likely because of the long flexible linker between the photocobilin and DGC domains. To understand the photo-activation process in more detail, we performed small-angle X-ray scattering (SAXS) measurements using the ‘dark’ and ‘light’ states of the full-length protein (SASBDB SASDUS3 and SASDUT3 respectively). The results show a significant difference in the scattering signal between the dark and light state (Fig. 9a−c). From the SAXS signals the radius of gyration Rg and the extrapolated intensity at q → 0 I(0) can be extracted. Upon illumination Rg increases from 42.1 Å (dark) to 52.2 Å (light), thus revealing an expansion of the protein, while I(0) remains almost unchanged (1.03 × 10³ and 1.08 × 10³ for dark and light state, respectively), indicating that the protein remains in its dimeric form. Fourier transform of the SAXS signals produces the signal distribution function p(r), which reveals a transition from a compact single peaked distribution for the dark state to a larger multidomain conformation for the light state (Fig. 9b). Low-resolution ab initio modelling based on the p(r) distributions shows that the AlphaFold2²⁵ prediction for AbDPcob is compatible with the dark conformation in solution, while the light state is characterised by an extended space arrangement largely different with respect to the dark state (Fig. 9c and Supplementary Fig. 23). Molecular dynamics simulations were conducted to compare dynamics of AbDPcob in the light and dark states (Supplementary Figs. 7–9). AbDPcob showed larger conformational change in the light state compared to that in the dark state indicated by structural deviation (RMSD) from the same starting structure (Supplementary Fig. 8). Specifically, the Pcob domain stays more stable than the DGC domain in both light and dark state. The DGC region was observed to show higher flexibility in the light state particularly at the interface with the Pcob region (Supplementary Fig. 7). Further analysis revealed an obvious opposite movement of the Pcob and DGC domains in the light state but not in the dark state (Fig. 9d, Supplementary Fig. 23 and Supplementary Movie 1 and 2). This indicates that photoactivation of AbDPcob is modulated by local protein conformation changes.

**Fig. 9: Light induced structural changes in AbDPcob.**

Although a clear photoactivation role has been established for the photocobilin domain fused to the DGC enzyme domain, the function of the standalone photocobilin photoreceptors is less clear. To study this aspect, we measured the activity of the isolated AbDGC domain upon mixing with an equal molar ratio of either of the two photocobilin proteins, isolated AdoCbl-bound forms of AbPcob or SasPcob (with and without bound BV) (Fig. 8d). Similar to the previous findings, the light-adapted states of the B₁₂-bound AbPcob and the holo SasPcob (i.e., both AdoCbl and BV bound) activate AbDGC catalysis. The increase in enzyme activity is dependent on the concentration of the photocobilin protein (Fig. 8d), suggesting a potential role for protein-protein interactions in the activation process. Surprisingly, in the absence of BV, the AdoCbl-bound SasPcob dark state activates AbDGC activity, whereas the light state appears to inhibit activity (Fig. 8d). Further evidence that the two proteins interact is provided by size-exclusion chromatography multi-angle light scattering measurements, which show dissociation of the AbDGC dimer into monomers upon addition of the photocobilin domain (AbPcob). (Supplementary Fig. 2). Higher concentrations of the photocobilin domain give rise to the appearance of a higher molecular weight species, which is likely to represent a heterodimer between the photocobilin and the AbDGC protein. Taken together, these results suggest that the standalone photocobilin photoreceptors can also regulate protein function through protein:protein interactions (PPIs), a mechanism that could potentially influence a wide range of metabolic pathways.

A wider role for photoglobins in cellular processes

Previous bioinformatics approaches have shown that photoglobin domains can be fused to several other types of functional domain and have proposed that coupled photoglobin and B₁₂-binding domains (i.e., photocobilins) may be involved in the regulation of different enzymes and transcription factors¹³. We have now shown that the photocobilins have the ability to regulate enzyme activity through PPIs. As such, we have investigated potential PPI networks to explore possible biological functions in cell metabolism (Supplementary Fig. 24). In Saccharothrix species, SasPcob potentially interacts with STAS (Sulphate Transporter and AntiSigma factor antagonist) domain, Stage II sporulation protein E (SpoIIE) and Sigma factor PP2C-like phosphatases (Supplementary Figs. 24 and 25a), suggesting a putative role in regulation of the cell stress response. Other photocobilins were frequently involved in one carbon pool by folate, amino acid metabolism and purine biosynthesis, suggesting potentially broad participation in cell metabolism (Supplementary Figs. 24 and 25a). Sequence similarity networks shows there are a wide range of photocobilin photoreceptor proteins with a high similarity of overall sequence, structure, motifs and domain architecture, either fused to other enzyme domains or as standalone proteins (Supplementary Figs. 26−30). A comparison of our SasPcob structure with other photocobilin proteins (predicted structures obtained from the recently published Alphafold2 database²⁶), shows they can be divided into three main groups depending on the location of the AdoCbl-binding domain at either the N- or C-terminus, and whether they contain the additional output domain in the fused proteins (Supplementary Figs. 25 and 31). The majority of the predicted structures align well with SasPcob (average RMSD = 1.27 Å). Some of these putative photocobilin proteins have variable lengths of the linker region between the AdoCbl- and BV-binding domains, which may affect the arrangement of the overall scaffold, while a small number of the fusion proteins lack a complete photocobilin domain. As the Alphafold2 structural models only show the monomeric form of the protein it is likely that these structures may differ in the higher order oligomeric form of the protein.

Conclusions

In conclusion, we have identified and characterised a new sub-family of AdoCbl-dependent photoreceptors that uniquely use two different chromophores to expand the wavelength range of B₁₂ for sensing and responding to light. The close proximity of the B₁₂ and BV cofactors in the photocobilins allows interaction and ‘cross-talk’ between them, meaning excitation of BV triggers B₁₂ photochemistry. This leads to structural change in the photocobilin protein that can promote enzyme activity either through direct fusion to the enzyme domain or by protein-protein interactions. The photocobilins are likely to have multiple functions in regulating cell metabolism and provides further light-sensing roles for B₁₂ in biology. The unique coupling of B₁₂ and BV in these novel photoreceptor proteins now opens a window to their exploitation as new optogenetic components.

Methods

Protein expression and purification

The proteins were expressed and purified as previously described with slightly modifications²⁷. The potential photoreceptor DNA sequences were selected, codon optimised and synthesised by GeneArt® (ThermoFisher) company. Synthesised genes were sub-cloned into a pET21a (Novagen) vector with a C-terminal 6·His tag. The recombinant plasmid was transformed into E. coli BL21(DE3) for protein expression. Different IPTG concentrations and auto-induction LB medium were used to optimise soluble expression. The large-scale protein expression was carried out with auto-induction LB media (FormediumTM, glucose/lactose ratio 1:4) containing 50 μg/mL ampicillin. After 24 h incubation at 25 °C, cells were harvested by 10 min centrifugation at 6000 g, 4 °C. Harvested cells were resuspended in 20 mM HEPES pH 7.0, 500 mM NaCl, 25 mM imidazole (lysis buffer) supplemented with protease inhibitor cocktail and then lysed by a cell disruptor at 25 kpsi (Constant Systems). The cell lysate was centrifuged at 20,000 rpm (48,000 × g) for 1 h at 4 °C to remove cell debris. The supernatant was collected and loaded onto a HisTrap column (Cytiva). After washing with cell lysis buffer, bound protein was eluted with 20 mM HEPES pH 7.0, 500 mM NaCl, and 250 mM imidazole. The peak fractions were collected and incubated with ligands (either AdoCbl, MeCbl or BV) for at least 2 h at 4 °C under dark conditions. The sample was then loaded on to a size exclusion column (HiLoad 16/600 Superdex 200) to remove free ligands and further purification. Absorbance spectra were used to confirm ligand binding and protein fractions with ligand bound were collected for further experiments. The CarH protein was expressed and purified for photoproduct determination (Supplementary Figs. 10−21) as described²⁷.

Crystallisation, data collection, and structure determination

Purified proteins were exchanged into 20 mM HEPES pH 8.0, 150 mM NaCl and concentrated to 20 mg/ml. Crystallisation was performed in the dark using the sitting drop vapour diffusion technique (200 nL crystallisation reagent mixed with 200 nL protein). The preparation of the dark sample was conducted in the dark with dimmed red light. In order to achieve full conversion to the final light-adapted state, the protein samples were exposed to a 530 nm LED light for a duration of 5 min. Absorbance spectral analysis was performed to ensure complete and effective illumination. The SasPcob dark crystals were obtained from LMB crystallisation screen B7 with 38% v/v 1,4-dioxane. The SasPcob light crystals were obtained from 0.2 M Potassium citrate tribasic monohydrate with 20% w/v PEG 3350. The AbPcob crystals were obtained from 8% w/v PEG 20000, 8% v/v PEG 550 MME, 0.1 M sodium acetate, pH 5.5, 0.2 M potassium thiocyanate. Crystals were cryo- protected by the addition of 20% PEG 200 to the reservoir solution and flash frozen in liquid nitrogen. Individual datasets were collected from single crystals at beamlines i03 (Diamond Light Source). All data were indexed, scaled and subsequently integrated with Xia2 Dials. Structure determination was initially performed by molecular replacement in Phaser²⁸ using a search model generated by Alphafold2²⁵. A combination of automated and manual rebuilding and refinement in Refmac²⁹ and COOT³⁰ were used to produce the refined models. Chromophore ligands were obtained from Refmac monomer library by 3 letter code and refined using COOT³⁰ and Refmac²⁹. Validation with both Molprobity³¹ and PDB_REDO³² were integrated into the iterative rebuild process. Complete data collection and refinement statistics are available in Table 1. The atomic coordinates and experimental data have been deposited in the Protein Data Bank (www.pdb.org). All figures were made using open-source PyMOL 2.5 software.

LED illumination and absorbance spectroscopy

Absorbance spectra were collected using a Cary 60 spectrophotometer (Agilent Technologies). All measurements were carried out under dimmed red light. The dark spectrum was collected prior to any illumination. A TDS3032C 300 MHz Digital Phosphor Oscilloscope (Tektronix) and TGP110 10 MHz Pulse Generator with Delay (Thurlby Thandar Instruments) were used to generate a 100 ms LED (Thorlabs Inc.) pulse at either 530 nm (green light) or 660 nm (red light). After each LED pulse, the spectrum was collected until there were no further significant changes. The difference spectra were obtained by subtracting the dark spectrum from the illuminated spectrum. Data were normalised and plotted using Origin 9.0 software (OriginLab, Northampton, MA).

B₁₂ and BV binding measurements

The binding ratio of B₁₂ and BV ligands was calculated based on their extinction coefficient. AdoCbl, 8.0 mM⁻¹•cm⁻¹ at 540 nm³³. MeCbl, 7.7 mM⁻¹•cm⁻¹ at 540 nm³⁴. BV, 90 mM⁻¹•cm⁻¹ at 700 nm³⁵. The protein concentration was determined by utilising the ProtParam tool³⁶ to calculate its theoretical extinction coefficient derived from its protein sequence. SasPcob, 55.46 mM⁻¹•cm⁻¹ at 280 nm. AbDPcob, 63.91 mM⁻¹•cm⁻¹ at 280 nm. The binding constant was measured by monitoring the spectral changes at increasing protein concentrations. The absorbance change at 540 nm was plotted for B₁₂ binding and the absorbance change at 700 nm was plotted for BV binding. With tight binding ligand, the binding constant (K_d) is much lower than ligand concentration used in the titration assay, tight binding equation (Eq. 1) should be used to get a more accurate K_d value. BV binding data were fitted to the tight binding equation (Eq. 1) and AdoCbl binding data to apparent binding equation (Eq. 2) to obtain the K_d value using Origin 9.0 software (OriginLab, Northampton, MA).

$$\left[{EL}\right]=\frac{\left(\left[E\right]+\left[L\right]+{K}_{d}\right)-\sqrt{{\left(\left[E\right]+\left[L\right]+{K}_{d}\right)}^{2}-4\left[E\right]\left[L\right]}}{2\left[L\right]}+c$$

(1)

$$[{EL}]=\frac{\left[E\right]\times [L]}{{K}_{d}+[E]}+c$$

(2)

where [EL] is the concentration of the enzyme and ligand complex formed, [E] is the enzyme concentration and [L] is the concentration of the ligand, c is the constant.

Diguanylate cyclase activity assay

The diguanylate cyclase activity was determined by using both liquid chromatography–mass spectrometry (LC-MS) and a pyrophosphate assay. For LC-MS, the reaction was prepared with 10 µM enzyme, 200 µM GTP in 20 mM HEPES buffer, pH 6.8, 150 mM NaCl, 10 mM MgCl₂.The reaction was carried out at 30 °C for 5 min, then deactivated at 95 °C for 5 min. The dark state reaction was carried out in a black tube and the light state reaction in a transparent tube after illumination with white light. All samples were then cooled down on ice and centrifuged for 15 min at 15,000 rpm. Samples were diluted 50 times with HPLC grade water and transferred to LC-MS glass vials for quantification on a Waters ACQUITY Xevo TQ-S UPLC–MS/MS (Waters Corporation) under negative mode (ESI-). The analysis was carried out using an Acquity UPLC HSS T3 column (1.7 µm, 50 × 2.1 mm) with an optimised gradient programme. The mobile phases were as follows; A: 10 mM ammonium acetate in water contains 0.1% acetate acid (v/v); B: methanol; flow rate: 0.6 mL/min; column temperature: 30 °C. The gradient starts from 97% A and hold for 0.5 min then decreased to 2% in 0.3 min, hold the 98% B for 0.4 min then returned to 97% A in 0.1 min and equilibrated for 0.7 min for waiting another injection. The detection of cyclic-di-GMP was optimised by employing the multiple reaction monitoring transition of 690.9 > 152.0 to quantify the yield of the reaction. The mass spectrometer parameters were as follows: source desolvation temperature of 600 °C and source ion block temperature of 150 °C were used; Nitrogen (≥95%) desolvation gas was set at 1000 L/h, and nebuliser gas at 7.00 Bar; Argon (zero grade) collision gas flow was set at 0.15 mL/min. The system was trained with a series of standard solutions of cyclic-di-GMP and the limit of detection limit was determined. The quantification standard curve was obtained ranging from 6 nM to 20 µM.

The EnzCheck pyrophosphate assay kit (ThermoFisher) was used to follow the kinetics of diguanylate cyclase activity. The reaction was carried out with 1−10 µM enzyme, 0−100 µM GTP in 20 mM HEPES buffer, pH 6.8, 150 mM NaCl, 10 mM MgCl₂. The reaction mixture was incubated in the dark or by illumination with a 530 nm LED for 3 min at 30 °C, prior to the addition of GTP to initiate the reaction. Pyrophosphate standard (0−60 μM Na₄P₂O₇) in the same buffer condition was used to generate the standard curve for accurately quantifying pyrophosphate concentration. The pyrophosphate produced was measured and quantified and then converted to cyclic-di-GMP production rate (two molecules of pyrophosphate equals to one molecule of cyclic-di-GMP). All data were collected in triplicate and plotted against GTP concentration. The K_m was obtained by fitting data into the Michaelis-Menten equation (Eq. 3).

$$v=\frac{{V}_{\max }\times [S]}{{K}_{m}+[S]}$$

(3)

SAXS data collection and analysis

Static X-ray scattering data were collected at 20 °C at the BM29 BIOSAXS beamline of the ESRF Synchrotron (Grenoble, France). X-ray solution scattering signals in the SAXS region (q = 0.0025−0.5 Å⁻¹) were collected on AbDPcob at 5 mg/mL concentration using a monochromatic X-ray beam (double multilayer monochromator) centred at 12.5 keV and a Pilatus3 2 M (Dectris) detector. Two datasets were collected, before and after irradiation of the AbDPcob solution with a LED light source at 530 nm for 10 min. For each dataset, 10 X-ray scattering signals (each registered with 1 s of X-ray exposure) were collected with 10 analogous signals of the buffer before and after each protein measurement. Each two-dimensional pattern was converted to a one-dimensional scattering profile by azimuthal integration. Corresponding scattering profiles were averaged and protein signals were obtained by subtraction of the buffer signal. The distance distribution functions p(r) were computed using GNOM³⁷, and the Rg and I(0) parameters were determined from the reduced data using routines from the ATSAS suite³⁸. Low-resolution molecular envelopes were computed using DAMMIF³⁷.

Molecular dynamics simulations of AbDPcob and AbPcob in light and dark state

The full-length AbDPcob and AbPcob was modelled using AlphaFold2²⁵ predicted and solved crystal structure respectively. The AdoCbl and cobalt was placed in AbDPcob by taking the coordinate in the crystal structure of AbPcob after structure alignment to Pcob domain. OHCbl was created by a simple mutation of the upper ligand in PyMol 2.5. We simulate the dynamics of AbDPcob and AbPcob in light and dark state by including different upper ligand into the protein: AdoCbl for dark state and OHCbl for light state. The input files for the calculation of parameters for AdoCbl and OHCbl were generated by MCPB.py protocol³⁹ in AmberTools 22^40,41 package. The starting structure of AdoCbl from the crystal structure was used as the starting geometry and the starting structure of OHCbl was created by replacing the adenosyl group with OH group in AdoCbl. Both ligands were first optimised by B3LYP/6-31 G*⁴² in Gaussian 09⁴³ and the optimised structure was used for calculating point charges and force constants. The protonation state of protein residues were calculated by PROPKA⁴⁴ in the presence of AdoCbl/OHCbl. AMBER99SB force field⁴⁵ was applied to model protein and ions. The complex structure was solvated by TIP3P modelled water molecules⁴⁶ in cubic box, keeping at least 14 Å between any atoms of protein and the boundary of the box. Counterions (19 Na⁺ ions for truncated AbPcob and 58 Na⁺ ions for full-length AbDPcob) were added to neutralise the system. MD simulations were carried out by Gromacs version 5.0⁴⁷. We use the same protocol for the simulation for the protein in light and dark state.

The system was first minimised to remove possible clashes after adding hydrogens to the protein, followed by a heating step to 300 K under NVT ensemble. The initial velocities were generated to maintain a Maxwell−Boltzmann distribution at the desired temperature, 300 K. The stepwise equilibration steps are conducted in order to make sure the structure gradually relaxed in the solvent environment: firstly, protein-complex were restrained to minimise/equilibrate (100 ps) water and ions; secondly, heavy atoms of protein and AdoCbl/OHCbl-Cobalt were restrained to minimise/equilibrate (100 ps) protein hydrogens; next main chain of protein and heavy atoms of AdoCbl/OHCbl and Cobalt were restrained to minimise/equilibrate (2 ns) protein side chains; then, protein Cα atoms and heavy atoms of AdoCbl/OHCbl and Cobalt were restrained to minimise/equilibrate (2 ns) main chain of protein; in last step, all restraints were remove to equilibrate for 2 ns. Harmonic restraint was applied before full relaxation of structures with a force constant of 1000 kJ/mol/nm². Production was run at 300 K by NPT ensemble for 1500 ns based on the equilibrated structure (500 ns for AbPcob). V-rescale thermostat algorithm⁴⁸ was used to control the temperature with a response time 1.0 ps. The pressure was kept at 1.0 bar using the Parrinello-Rahman pressure coupling scheme⁴⁹ with a response time 1.0 ps. Hydrogen atoms were constrained during the production by LINCS⁵⁰. The cut-off was set to 12 Å for electrostatic interactions calculation. Additional sampling was carried out on AbDPcob using three parallel simulations as follows: 50 × 1 ns cycles of simulated annealing (700 ps at 300 K, 100 ps heating to 400 ps, 100 ps at 400 K, 100 cooling to 300 K) followed by an additional 350 ns of MD for each run, which was then used for analysis. The increased sampling is clear from the Cα RMSDs for these simulations (more details see supplementary information).

Ensemble docking of BV to AbPcob

The AbPcob conformations in dark state after MD simulation was used for BV docking analysis. AbPcob dark conformations were clustered based on RSMD values of BV binding region. Gromacs gromos⁵¹ method was used for clustering simulated structure at 1.5 Å cut-off. 38 clusters were obtained with RMSD cut-off of 1.5 Å for two structures to be neighbour. Autodockvina⁵² was used to dock BV into clustered AbPcob structure with a grid box size of 18 Å × 18 Å × 18 Å. All docked binding poses were ranked by binding affinity. Structures with a value below −7.0 kcal/mol were selected for further docking analysis. To get more detailed binding possibilities, the search exhaustiveness was set to 72 to rerun molecular docking with selected structures. The docking results were used to analyse the possible binding pose of BV to AbPcob. A similar procedure was used for the MD simulations of AbDPcob following simulated annealing, with and RMSD cut-off of 1.0 Å, resulting in 100 clusters (15 for one binding pocket, 85 for the other) to ensure an exhaustive conformational sampling during docking, and an exhaustiveness of 25.

Size exclusion chromatography-multi-angle light scattering (SEC-MALS)

SEC-MALS was used to determine the oligomeric state of the target proteins and to probe any protein-protein interactions, under different conditions. An Agilent G7110B HPLC pump, degasser and autoinjector (Agilient, Santa Clara, USA) was used to auto load the samples (50 µL each run, 1 to 10 mg/mL). Superdex 200 10/300 GL column was used for chromatographic separations. MiniDAWN TREOS MALS detector and Optilab rEX refractive index metre (Wyatt, Santa Barbara, USA) were used to collect light scattering signals. The flow rate was set at 1 mL min⁻¹ with a mobile phase of 20 mM HEPES, pH 6.8, 150 mM NaCl buffer. Samples were preincubated in dark for 30 min or with 530 nm LED for 5 min. All results were processed according to referenced protocol⁵³. Peak alignment, band-broadening correction and normalisation procedures were performed by selecting the central 50% region of the peaks. Raw data were exported and plotted using Origin 9.0 software (OriginLab, Northampton, MA).

Genomic contextual and protein-protein interaction-based enrichment analysis

A computational gene neighbourhood analysis was performed using Genomic enzymology tools⁵⁴. Briefly, a sequence similarity network (SSN) was constructed to facilitate the assignment of function for different proteins. Then, Genome Neighbourhood Networks (GNNs) were constructed based on SSN to provide statistical analysis of neighbouring Pfam families. The frequency of common domains identified in the GNN was analysed and visualised by R and R package ggplot2. As 506 sequences belong to divergent species, four species (Saccharothrix syringae, Streptomyces sp. M41, Micromonospora yangpuensis and Magnetospirillum magnetotacticum MS-1) that possess more than one hit among 506 were selected for further analysis. In each species, the sequence was submitted to the STRING database⁵⁵ for the construction of protein-protein interaction (PPI) network using two shells (20 interactors and 5 interactors) with medium confidence (0.400). Enrichment analyses, including Gene ontology, Local network cluster (STRING), reactome pathways, protein domains, and features (Pfam and InterPro) were performed based on gene lists from the PPI network. Enrichment results were visualised using R and R package ggplot2 and ggpubr.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The atomic coordinates and experimental data generated in this study have been deposited in the Protein Data Bank (www.pdb.org) under accession code: 8J2W, 8J2X, 8J2Y. Atomic coordinates referenced in the main text can be found through 1B33, 3MWN. SAXS data has been deposited in the small Angle Scattering Biological Data Bank (www.sasbdb.org) under accession codes SASDUS3 and SASDUT3. LCMS data has been deposited on Figshare (Jeffreys, Laura [2024]). Photoproduct determination - LCMS. Figshare. Journal contribution. https://doi.org/10.6084/m9.figshare.25219721. LCMS data has been deposited on Figshare (Jeffreys, Laura [2024]). Photoproduct determination - NMR. Figshare. Journal contribution. https://doi.org/10.6084/m9.figshare.25219832). MD parameters for AdoCbl and OHCbl have been deposited on Figshare (https://doi.org/10.6084/m9.figshare.25226480). Initial and final structures from MD simulations have been deposited on Figshare (https://doi.org/10.6084/m9.figshare.25226549). All other source data are provided as supplementary data files. Source data are provided in this paper. Source data are provided with this paper.

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Acknowledgements

The work was funded by the Engineering and Physical Sciences Research Council International Centre-to-Centre grant no EP/S030336/1. We thank Diamond Light Source beamlines I03 & I04 (proposal numbers mx2447-65, 87, 89). This work was also supported by National Natural Science Foundation of China (No. 31870855) and NUDT Research Program (22-ZZCX-039, 22-02-15). The authors also thank the ESRF (proposal MX-2398) for beamtime and the staff of beamline BM29 for assistance with data collection. An ANR grant (PhotoGene, ANR-21-CE11-0036-01) awarded to GS supported the research reported in this paper. The IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). The authors would like to gratefully acknowledge the training and use of mass spectrometry instrumentation provided by Professor Perdita Barran’s group at the Michael Barber centre for collaborative mass spectrometry (MBCCMS) based at the University of Manchester.

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Manchester Institute of Biotechnology and Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK
Shaowei Zhang, Laura N. Jeffreys, Harshwardhan Poddar, Yuqi Yu, Kaylee Patel, Linus O. Johannissen, Matthew J. Cliff, Cunyu Yan, Michiyo Sakuma, Colin W. Levy, David Leys, Derren J. Heyes & Nigel S. Scrutton
Department of Biology and Chemistry, College of Sciences, National University of Defense Technology, Changsha, China
Shaowei Zhang, Chuanyang Liu & Lingyun Zhu
Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France
Giorgio Schirò & Martin Weik

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Shaowei Zhang
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Contributions

N.S.S., D.J.H., D.L. and S.Z. initiated and coordinated the project. S.Z., L.N.J., D.J.H., N.S.S. and D.L. designed experiments, analysed data and wrote the manuscript with contributions from other authors. S.Z. produced and crystallised the proteins. H.P., M.S. and C.W.L. helped with protein crystallisation and data collection. S.Z. processed diffraction data and solved the structures. Y.Y., L.O.J. and S.Z. performed the docking and molecular dynamics simulations. C.L. and L.Z. carried out bioinformatics analysis of Pcob proteins. K.P., L.N.J. and M.J.C. assisted with SasPcob characterisation. L.N.J. and C.Y. performed mass spectrometry analysis. L.N.J. and M.J.C. performed all NMR experiments. G.S. and M.W. performed SAXS measurements. N.S.S., D.J.H. and D.L. advised on all aspects. All authors discussed the results and commented on the manuscript.

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Correspondence to Shaowei Zhang, Derren J. Heyes or Nigel S. Scrutton.

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Zhang, S., Jeffreys, L.N., Poddar, H. et al. Photocobilins integrate B₁₂ and bilin photochemistry for enzyme control. Nat Commun 15, 2740 (2024). https://doi.org/10.1038/s41467-024-46995-1

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Received: 18 May 2023
Accepted: 17 March 2024
Published: 28 March 2024
DOI: https://doi.org/10.1038/s41467-024-46995-1

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