Structural and Functional Analysis of a Prokaryotic (6-4) Photolyase from the Aquatic Pathogen Vibrio Cholerae

Photolyases are ﬂ avoproteins, which are able to repair UV-induced DNA lesions in a light-dependent manner. According to their substrate, they can be distinguished as CPD-and (6-4) photolyases. While CPD-photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6-4) photolyases catalyze the repair of the less prominent (6-4) photoproduct. The subgroup of prokaryotic (6-4) photolyases/FeS-BCP is one of the most ancient types of ﬂ a-voproteins in the ubiquitously occurring photolyase & cryp-tochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6-4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential ﬂ avin adenine dinucleotide as well as an elongated linker region between the N-terminal α / β -domain and the C-terminal all-α - helical domain. Furthermore, they can harbor an additional short subdomain, located at the C-terminus, with a binding site for a [4Fe-4S] cluster. So far, two crystal structures of prokaryotic (6-4) photolyases have been reported. Within this study, we present the high-resolution structure of the prokar-yotic (6-4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA-repair

So far, almost all canonical photolyases were described as monomeric proteins in solution with apparent molecular masses of 45-60 kDa, a bilobal domain architecture with an N-terminal α/β-domain and a C-terminal all-α-helical domain, and binding sites for two chromophores (14).The first is flavin adenine dinucleotide (FAD), which is bound non-covalently in an unconventional U-shaped conformation and is essential for light-dependent DNA-repair activity.The second chromophore (an antenna chromophore), is also non-covalently attached and can be either a second flavin species e.g.FAD (15) or flavin mononucleotide (FMN) (16) or, more commonly, a tetrahydrofolate-(methenyltetrahydrofolate: MTHF) (17) or deazariboflavin-derivative (8hydroxy-7,8-didemethyl-5-deazariboflavin, 8-HDF) (18).This antenna serves as a light-harvesting chromophore and is bound by the N-terminal domain, embedded within a characteristic Rossmann fold.Besides their biological ability to repair UV-induced DNA lesions, a process commonly referred as photoreactivation, photolyases are also able to undergo a second blue-light-triggered reaction.Within this process (photoreduction), the flavin chromophore is either fully oxidized (FAD ox ) or semiquinoid, radical species (FADH • ) gets reduced to the fully reduced (FADH − ) state, which is the only catalytically active redox state of FAD within photolyases (19).This light-triggered intraprotein electron transfer (ET) process often takes place via a tryptophan triad, a highly conserved structural motif within canonical photolyases (20).
In contrast, the subgroup of prokaryotic (6-4) photolyases possesses a few unique properties in comparison to all other classes of photolyases.While sharing their typical bilobal domain architecture, they exclusively incorporate a lumazine derivative (6,7-dimethyl-8-ribityllumazine, DLZ) as antenna chromophore and bind additionally to an iron-sulfur cluster [4Fe-4S] besides their catalytic FAD moiety.The [4Fe-4S] cluster is located in the short additional roof-like subdomain at the C-terminus (6).Based on this additional cofactor, this subgroup was initially labeled as FeS-BCP (Fe-S containing bacterial cryptochromes and photolyases).However, later phylogenetic analysis reveals that not all members of this subgroup possess a [4Fe-4S] cluster and might own only a DNA repair activity for (6-4)PP lesions and function less as cryptochromes.Therefore, we will refer to this subgroup as prokaryotic (6-4) photolyases.On a phylogenetic level, these photolyases are only distantly related to other PCSf subgroups and were postulated to be the most ancient subgroup of PHLs (21).However, this notion might be tackled by the recent discovery of NewPHLs, with a more simplified domain architecture but retained CPD repair activity (9).In general, the roof-like subdomain of prokaryotic  photolyases shares a structural homology with the C-terminal domain of PriL from eukaryotic and archeal primases, which also contains a [4Fe-4S] cluster (22).So far, two crystal structures of prokaryotic (6-4) photolyase were successfully solved, namely, PhrB from the soil bacterium Agrobacterium fabrum (PDB entry: 4DJA) (6) and RsCryB, originated from the purple bacterium Rhodobacter sphaeroides (PDB entry: 3ZXS) (8).
Previous sequencing of the γ-proteobacterium Vibrio cholerae (strain El Tor N16961, from now on referred as V. cholerae) revealed that its genome harbors three genes belonging to the PCSf (23).Initial biochemical analysis by Worthington et al. indicated that one of these genes is a canonical CPD-photolyase (VcPhr), and the other two were referred as cryptochromes (namely, VcCRY1 & VcCRY2) (24).Further studies revealed that VcCRY1 is a member of the Cry-DASH subgroup (25), while a current bioinformatic analysis of the PCSf showed that VcCRY2 is a member of the so called NewCRY cluster, a recently discovered subgroup of cryptochromes with unique properties, e.g., an incompletely conserved tryptophan triad for electron transfer during photoreduction (9).However, a hypothetical gene (VCA0809) of the V. cholerae strain N16961 was identified as a potential photolyase/cryptochrome gene due to its strong upregulation upon blue-light illumination (26).VCA0809 could be grouped within the FeS-BCP subgroup phylogenetically, and therefore the protein was identified as a prokaryotic (6-4)-photolyase, Vc .Furthermore, this study performed an initial characterization in regards to spectroscopic properties and DNA-repair activity of Vc(6-4) (27).Here, we present the highresolution crystal structure of Vc(6-4) and complete the biochemical and photochemical characterization of this (6-4)photolyase.

MATERIALS AND METHODS
Plasmid constructs.The Vc(6-4)-coding plasmid was obtained as a synthetic gene from BioCat (Heidelberg, Germany).The construct is based on the pET28a(+)-vector and codon-optimized for heterologous gene expression in E. coli.Vc  is fused to an N-terminal His 6 -tag for initial purification via IMAC.Sequence information of Vc(6-4) is deposited under UniProt-ID Q9KLD7 (Gene: VCA0809, V. cholerae (strain El Tor N16961)).
Recombinant expression and purification of Vc(6-4).The expression vector pET28-Vc(6-4) was transformed into chemically competent E. coli BL21(DE3) Gold cells (Stratagene).For protein expression, bacteria were grown in LB-medium supplemented with kanamycin at 37°C to an OD600 of 0.6-0.8.Thereafter, expression was induced by addition of 20 μM isopropyl-β-D-thiogalactopyranoside (IPTG).Cells were shaken at 18°C for 4 h, collected by centrifugation, and resuspended in buffer A (50 mM NaH 2 PO 4 , 300 mM NaCl, pH = 8).Cell disruption was performed with a French Pressure cell (Aminco) at a constant pressure of 1000 psi.Subsequent to cell disruption, cell debris was pelleted by centrifugation at 4°C and 15.000 rpm for 60 min.Vc(6-4) was purified by immobilized metal ion affinity chromatography (IMAC) as the initial purification step; additionally, heparin affinity chromatography was performed to remove potential DNA contaminants.For IMAC, the supernatant was sterile-filtered and applied to a buffer A preequilibrated column packed with Ni 2+ -NTA agarose matrix (Qiagen).The column was washed by four column volumes (CVs) of 10% buffer B (50 mM NaH 2 PO 4 , 300 mM NaCl, 500 mM imidazole, pH 8), and thereafter Vc(6-4) was eluted with a final imidazole concentration of 250 mM (50% buffer B).For heparin affinity chromatography, protein fractions were pooled and diluted 1:4 with buffer A and applied to a heparin column (HiTrap heparin, GE Healthcare).The column was washed with four CVs buffer A and Vc(6-4) was eluted with 1 M sodium chloride (50% buffer C: 50 mM NaH 2 PO 4 , 2 M NaCl, pH 8).For protein crystallization, a final purification step, using size exclusion chromatography (SEC), was performed.For that, a HiLoad Superdex 26/600 (GE Healthcare) column was used that was equilibrated in 20 mM Tris, 200 mM NaCl, 5% glycerol, pH 7.8 at 4°C.
The result of the Vc(6-4) purification was monitored by SDS-PAGE (Figure S1).After the final purification step, Vc(6-4) was obtained with an overall yield of 10 mg per liter expression culture with a purity >95%.
Analytical size exclusion chromatography of Vc .The oligomeric state of Vc  in solution was determined via analytical size exclusion chromatography.A Superdex 75 Increase 10/300 (GE Healthcare) column and a protein concentration of 1 mg L −1 were used, and SEC was performed in 20 mM HEPES, 100 mM NaCl, 5% glycerol, pH 8 at 4°C.The oligomeric state was determined by calibration with standard proteins (conalbumin, ovalbumin, and ribonuclease A) according to the manufacturer's manual.
Characterization of Vc(6-4) by UV-Vis spectroscopy.All spectroscopic measurements and in vitro activity assays for recombinant Vc(6-4) were performed with a V-660 photometer (JASCO) at 10°C.Initial spectroscopic characterization was performed after heparin affinity chromatography in buffer A; for in vitro photoreduction of protein bound FAD, 25 mM dithiothreitol (DTT) was added to the protein solution.The sample was incubated for 5 min in darkness and afterward illuminated with a blue light LED with a maximum emission wavelength of 455 nm (ILS, Intelligent LED Solutions) at 4 cm distance (light intensity: 5.9 mW•cm −2 , i.e. 224.41 μmol•m −2 •s −1 ).Spectra were recorded at different time points, ranging from 1 min to 75 min.
Crystallization and structure determination of Vc(6-4).Vc(6-4) was concentrated, using a 50 kDa cut-off centrifugal filter (Amicon, Merck, Germany) to a final concentration of 6.25 mg mL −1 .Protein concentration was determined by the Bradford-assay (29).Initial 96-well format crystallization screening was performed with a Cartesian robot system and different commercially available crystallization screens using a sitting drop vapor diffusion method at 20 °C.Yellowish protein crystals were obtained after 14 days in the JCSG Core IV Suite (30) (Qiagen), within a condition containing 0.1 mM sodium chloride, 0.1 Tris, 1.5 M ammonium sulfate, pH 8.For cryoprotection, glycerol was added to a final concentration of 30%, and Vc(6-4) crystals were flashfrozen in liquid nitrogen.X-ray data were collected from a single crystal at 100 K at beamline X06SA (PXI) at the Swiss Light Source (SLS, Villigen, Switzerland).Vc(6-4) was crystallized in the hexagonal space group P6 4 22 with unit cell parameters of a = b = 199.69Å, c = 77.00Å. Diffraction data were processed by XDS (31).The structure was solved via molecular replacement with ARP/wARP (32) and PHASER MR (33), using a homology model of Vc(6-4), derived from RsCryB (PDB entry: 3ZXS) (8).Further refinement was performed with a combination of phenix.refine(34), REFMAC5 (35) from the CCP4 software package (36) and COOT (37).Final refinement led to R-factors of 0.125/ 0.142 for R work /R free at an overall resolution of 1.65 Å.Data processing and refinement statistics are summarized in Table 1.Further structural analysis and figures were carried out with the PyMOL software.The crystal structure of Vc(6-4) is deposited in the protein data bank (PDB entry: 8A1H).
Bioinformatic analysis of prokaryotic (6-4) photolyases.For bioinformatic analysis in regard to conserved regions within prokaryotic (6-4) photolyases, a multiple sequence alignment with all entries of database entry PTHR38657 (>11 000 sequences of prokaryotic (6-4) PHLs & Crys) was performed with MAFFT (38).The resulting MSA was analyzed and visualized with the WebLOGO webserver (39).For visualization of the conserved region on a structural level, the ConSurf pipeline (40) was used and the level of conservation was mapped onto the structure of Vc .

RESULTS AND DISCUSSION
Expression, purification, and spectroscopic characterization of Vc  Independently from the study of Dikbas et al. (27), the identical gene from V. cholerae was identified as a FeS-BCP member by sequence similarity networks (9) and selected for further studies.After initial purification of Vc(6-4) by immobilized metal ion affinity chromatography (IMAC), an additional purification step was necessary via heparin affinity chromatography to remove DNA which is bound unspecifically to the photolyase.After this purification step, Vc(6-4) eluates as a brownish-yellowish protein.
Overexpression and purification were analyzed by SDS-PAGE (Figure S1), showing a strong band of purified Vc(6-4) with an apparent molecular mass of approximately 60 kDa, which fits to the calculated molecular mass of 61.6 kDa for Vc  fused to an N-terminal hexa-histidine tag.Initial absorption spectra after purification (Fig. 1a, black solid line) are characterized by linearly increasing absorption at 500-700 nm, caused solely by the [4Fe-4S] cluster, and major absorptions in the blue-light region with absorption peaks at 382, 420, and 440 nm and a shoulder at 475 nm.These absorption properties are similar to previous results of Dikbas et al. for Vc(6-4) (27) or other well-described prokaryotic (6-4) photolyases (8,41,42).The absolute maximum is at 420 nm and is mainly caused by the antenna chromophore DLZ and contributions by the [4Fe-4S]-cluster.
Vc(6-4) undergoes photoreduction upon blue-light illumination and in the presence of an external reduction agent (Fig. 1a), leading to the fully reduced species FADH − .During this process, the spectral signature of Vc(6-4) in the blue-light region simplifies and the absorption peaks at 382, 440, and 475 nm vanish after 75 min of blue-light illumination.The resulting spectrum (Fig. 1a, red line) shows only one broad peak at 417 nm, slightly blue-shifted in comparison to the former peak at 420 nm.During photoreduction, there is a small increase of absorption around 620 nm within the first minutes of illumination, caused by the transient formation of the semiquinone radical species FADH•.However, this absorption subsequently decreases, indicating that there is no major accumulation of FADH• during photoreduction.The corresponding light minus dark difference spectrum (see Fig. 1b) shows that the FAD chromophore in Vc(6-4) is photoreduced to the fully reduced FADH − state.After 75 min of blue-light illumination, the resulting difference spectrum shares the typical absorption pattern of protein-bound FAD in the oxidized state, and they are quite similar to previously published difference spectra of PhrB (21).Statistics for the highest-resolution shell are shown in parentheses.

Structural analysis of Vc(6-4)
The crystal structure of Vc(6-4) was solved via molecular replacement at a final resolution of 1.65 Å.For molecular replacement, the structure of RsCryB (PDB entry: 3ZXS) was used (8).
The final statistics of data collection, processing, and refinement procedure are summarized in Table 1.
The overall fold of Vc(6-4) shares the typical bilobal domain architecture of photolyases and cryptochromes (Fig. 2a), with an α/β-domain, located at the N-terminus (aa M1-V127), and an all-α-helical domain at the C-terminus.While the N-terminal domain is built up by four parallel β-strands and six α-helices, the catalytic C-terminal domain consists of 20 α-helices (aa R233-L516).Both domains are connected via an elongated linker-region (aa D128-N232) which is wrapped around the overall protein fold.In comparison to canonical PHLs or CRYs; Vc(6-4) or prokaryotic (6-4) photolyases, in general, own a small well-ordered C-terminal extension, which harbors the binding site of the [4Fe-4S] cluster, which is covalently linked to four conserved cysteine residues within these "roof-like" subdomain.
Like other PCSf members, Vc(6-4) shares the strongly basic groove around the FAD-binding site, which enables the binding of UV-damaged DNA and allows a direct contact of this substrate to the FAD chromophore within the active site.The overall electrostatic charge distribution (Fig. 2b) of this putative DNAbinding site is similar to the structures of RsCryB and PhrB and is mainly formed by the α-helices α 25 , α 28, and α 29 of the catalytic domain and the helices α 8 and α 9 of the linker region.However, this binding groove seems to be more sterically constricted in contrast to the eukaryotic (6-4) photolyases.A superimposition (Figure S3) with the cocrystal structure of the (6-4) photolyase of Drosophila melanogaster (Dm (6-4)) bound to dsDNA with a (6-4)PP lesion (PDB entry: 3CVV, root mean square deviation (RMSD): 5.0 Å for 1707 aligned atoms) shows several steric clashes along the DNA binding surface, when considering the DNA duplex following the 3 0 -end of the (6-4)PP.This indicates a different binding modus for the 3 0 -arm of the dsDNA substrate by prokaryotic (6-4) photolyases when compared to eukaryotic-type (6-4) photolyases.Notably, two of the seven sulfate ions, which are defined in the Vc(6-4) crystal structure, mimic positions of phosphate groups of the 5 0 -arm of the dsDNA with (6-4)PP lesion from the superimposed Dm (6-4) complex.The first sulfate corresponds to the intralesion phosphate of the (6-4)PP, whereas the second sulfate is close to the phosphate positions of the counter-strand of the (6-4)PP comprising the DNA strand at the −3 and − 4 positions.Likewise, one glycerol (total: 11) occupies the position of the 3 0 -ribose moiety of the (6-4)PP at the top of the active site and is bound between M378, Y431, and Y437.The corresponding tyrosines in PhrB from A. tumefaciens, Y424 and Y430, have been shown to affect the binding and repair of (6-4) lesion DNA when being replaced to phenylalanine (43).
Overall, Vc(6-4) harbors three different chromophores; the catalytic essential FAD; a lumazine-derivative DLZ, which serves as an antenna chromophore; and the additional [4Fe-4S]-cluster.The edge-to-edge distance between the catalytic essential FAD and the cluster is 18 Å, while the DLZ is located closer to the FAD, with an edge-to-edge distance of 12.6 Å (Fig. 2, lower panel).The electron transfer between the FAD chromophore and the bulk solvent (Fig. 2) consists of the proximal tyrosine pair Y398/Y402, the medial tryptophan W397, and the mostly surface-exposed W349 with centroid-to-centroid distances between 5.8 Å and 6.9 Å.

The FAD-binding site & active centre of Vc(6-4)
The FAD-binding pocket is deeply buried within the all-α-helical domain, and its FAD is bound in a U-shaped conformation.In contrast to canonical PHLs or CRYs; only a single water, WAT33, is located above the N5 nitrogen atom of the isoalloxazine moiety of FAD (Fig. 3a, Figure S2) and forms H-bonds with the backbone carbonyl of tyrosine Y398, the N5 and the carbonyl group (O4) of FAD.
This water is structurally preserved in the RsCryB and PhrB structures and, given the static nature of the Vc(6-4) structure, represents currently the only candidate for a proton donor to the N5 nitrogen during the protonation of the FAD •-radical after photoreduction.The alternative, the closely located glutamic acid residue E410, is apparently protonated but faces away from the FAD ring system and H-bonds to the side chain of the surface exposed reside D395.In other PCSf groups than the prokaryotic Photochemistry and Photobiology, 2023, 99 1251 (6-4) photolyases, the H-bonding partner to the N5 atom is either an asparagine, aspartate, or cysteine.While asparagine is a hallmark of the other photolyase groups (e.g., EcCPDI: N378 (44), MmCPDII: N403 (45), PhrA: N380 (46), DsNewPHL: N188 (9)), the corresponding position is replaced by an aspartic acid (AtCry1: D396) (47) in plant cryptochromes, or, in the case of insects cryptochromes, by a cysteine (e.g., DmCRY: C416) (48).Interestingly, the asparagine suffices to drive lighttriggered photoreduction (FADox ➔FADH • ➔FADH¯) and protonation via an unidentified protonation pathway upon FADox ➔ FAD •-photoreduction, whereas aspartate leads to accumulation of the semiquinone state FADH • ; the biologically relevant signaling-state of plant cryptochromes.In contrast, the cysteine in direct proximity to the N5 ring atom arrests photoreduction in the anionic radical state FAD •-(49).In any case, one has to postulate that protonation of the FAD •-state in photolyases and cryptochromes to FADH • requires a conformational change for transient opening of a proton conductance pathway to the protein surface (50).In Vc(6-4), such a proton wire may involve the residues E410 and D395, but these residues are conserved in only about half of the prokaryotic (6-4) photolyases.
Otherwise, this catalytic FAD chromophore is attached noncovalently to the protein via in a H-bond network.N278 is forming a H-bond to the O2 functionality of the isoalloxazine, like the nearby located N3-atom to the backbone oxygen of D404.The adenine moiety of the chromophore forms H-bond with several water molecules and N413.The HHXXR-motif, which is conserved in the subgroup of prokaryotic  photolyases, is also present in Vc .The motif consists of H372, H373, I374, Q375, and R376 and is located closely to the putative DNA-binding site and the FAD chromophore.Previously published mutagenesis studies of PhrB and RsCryB revealed that the second histidine position (Vc(6-4): H373, PhrB: H366, RsCryB: H362) is crucial for DNA-repair activity (51).This residue is denoted as the first catalytic histidine, His 1 , in eukaryotic  photolyases and animal cryptochromes.The majority of theoretical models for the catalytic (6-4)PP repair, independently whether they require a single or two photons for repair, predicted this residue in the protonated state (for discussion refer to ( 52)).In the Vc(6-4) structure, H373 adopts apparently the deprotonated state because its side-chain packs onto a sodium ion coordinated to Q313 and the adenine of the FAD cofactor.However, upon substrate binding, this sodium ion should get displaced by the (6-4)PP moiety.The other histidines, H372 and R376, are present in many CPD photolyases.The latter one, R376, interacts with aspartic acid D404; this salt bridge close to the FAD is a conserved structural feature of all members of the PCSf and plays a crucial role during the photoreduction of the catalytic chromophore, as shown by by serial femtosecond crystallography (53).
Unlike canonical photolyases and cryptochromes, the conserved tryptophan triad is not present within prokaryotic  photolyases.The proximal position of the Trp-triad is occupied by a tyrosine Y398 in Vc(6-4) with a distance of 3.7 Å to the isoalloxazine ring of FAD.In general, a conserved pattern of tyrosines and tryptophans is responsible for the intramolecular electron transfer during photoreduction within these  photolyases.These are W349, W397, Y398, and Y402 in Vc(6-4), with Y398 and Y402 being proximal to the FAD and W349 distal and solvent-exposed.Their distinct roles for photoreduction were extensively investigated by mutagenesis studies for the previous described PhrB and RsCryB.These studies revealed that both tryptophans (Vc(6-4):W349, W397; RsCryB: W338, W386; PhrB: W342, and W390) are crucial for in vitro photoreduction (8,43).Furthermore, the work of Holub et al. for PhrB (54) and the studies of Geisselbrecht et al. for RsCryB (8) pointed out that the proximal Y398 is also crucial for light-triggered reduction within prokaryotic (6-4) photolyases.By replacing this residue with alanine within PhrB, photoreduction is completely blocked.Interestingly, the authors reported a complete loss of both chromophores, FAD and DLZ, by replacing this tyrosine with the more bulky tryptophan, which is otherwise able to be part of an intramolecular electron transfer chain (54).
The antenna binding site of Vc(6-4) Vc(6-4) binds the lumazine derivative DLZ as antenna chromophore.This molecule is part of the natural riboflavin biosynthesis pathway (55) and seems to be the general antenna of prokaryotic (6-4) photolyases.DLZ is known as a strong fluorophore, which is involved in bacterial luminescence as the chromophore in luciferase accessory proteins (56).With these photochemical properties and a distance of 12.6 Å to FAD (closest distance between the aromatic ring systems), DLZ is a plausible antenna chromophore for fluorescence resonance energy transfer toward the catalytic essential chromophore within the active site.The distance is comparable with the chromophore arrangement in RsCryB (13.2 Å) and PhrB (13.0 Å).
The binding site is located in the N-terminal domain, and the chromophore is noncovalently attached to the protein via multiple H-bond interactions (Fig. 3b).The N1-atom of the pteridine ring is forming a polar interaction with the nitrogen backbone atom of glycine G11, as well as the neighboring carbonyl O 2 .Close to this functionality, a water molecule is embedded within a H-bond network, consisting of backbone atoms of A33 and I9.Further H-bonds are built up between the O 4 and N 5 atoms of DLZ and the residues L35 and Q39.The residues D12, E38, Y41, and D106 serve as H-bond acceptors within polar interactions with the ribityl-moiety of the antenna chromophore.Additionally, the aromatic system of F52 is facing toward the pteridine ring, leading to a face-to-face π-stacking interaction with a distance of 4.2 Å. H44, whose position is conserved in about two-thirds of all sequences of prokaryotic (6-4) photolyases (6), forms van der Waals contacts with both methyl groups of DLZ and impedes the binding of more voluminous antenna chromophores due to steric reasons.Interestingly, these interactions with the antenna slightly differ in comparison to RsCryB.Within this cryptochrome, an additional water molecule is present, forming directly H-bonds with N 5 and O 4 atoms of DLZ.This interaction is overtaken by glutamine Q39 in Vc(6-4).However, this additional water is also absent in the PhrB structure, where the N 5 atom is not participating in any direct polar interaction.A ConSurf (57) analysis based on Vc  indicates that residues G11, D12, L35, E38, H44, and D106, which are directly involved in antenna binding, are highly conserved within the prokaryotic (6-4) photolyases (Fig. 4).
The C-terminal roof-like subdomain of Vc  The iron-sulfur cluster is embedded between the roof-like subdomain at the C-terminus (S434-L516) and the catalytic domain.The cubic cluster is clearly visible by electron density and is bound via four conserved cysteines (C357, C461, C445, and C448, Fig. 3c).However, roughly 30% of prokaryotic  photolyases are lacking the pattern of four cysteines.One bacterial (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus (Pm(6-4)) without the binding motif for the [4Fe-4S]-cluster was previously described (7).Pm(6-4) is binding FAD substoichiometrically and is capable to repair UV-induced (6-4) lesion within ssDNA, with drastically lower repair efficiency in comparison to regular prokaryotic  photolyases with a [4Fe-4S] cluster and only in the presence of Mg 2+ ions.These results indicate light-driven DNA repair without the direct participation of the cluster.The unusually low repair efficiency might be explainable due the low amount of catalytic active chromophore within Pm (6-4).However, the idea that the [4Fe-4S] cluster somehow enhances the repair efficiency of prokaryotic  photolyases is currently feasible as well.
The C-terminus around the cluster binding site shares a structural similarity with the large subunit of eukaryotic DNA primases (Figure S4).These ubiquitously occurring proteins are polymerases, which are capable of synthesizing short RNA primer, which are important during replication (58).The region of similarity includes the α-helices α 20 -α 25 of Vc(6-4) and the conserved pattern of cysteines for the coordination of the [4Fe-4S] cluster.The structural superimposition with the large subunit of the Saccharomyces cerevisiae primase (PDB entry 3LGB) was done for amino acids M355-C461 of Vc , with an RMSD of 3.2 Å for 492 aligned atoms.The cluster is almost bound at the exact same position, and two of the four cysteines which are involved in cluster binding within this primase are found at corresponding positions.
Although iron-sulfur clusters were often described for redox active cofactors (59), the exact biological function of the ironsulfur cluster within prokaryotic (6-4) photolyases is not completely cleared out yet.However, previously published experimental results showed that it is not possible to obtain soluble mutants without the iron-sulfur cluster (43,51).The removal of the cluster-binding cysteine or the removal of the roof-like subdomain around the [4Fe-4S]-binding site resulted in insoluble proteins.Therefore, it might be that the [4Fe-4S] cluster plays somehow an important role for the structural integrity of these (6-4) photolyases (43).Due to a lack of experimental evidence for a protein-mediated electron transfer from the iron-sulfur cluster to the FAD chromophore, it is assumed that the redox-silent cluster is solely important for structural integrity and stability.Due to the close location of the cluster to the putative DNAbinding groove, the cluster might also affect the binding mode of DNA.
Mg 2+ promotes in vitro DNA-repair by Vc  We performed DNA-repair assays to investigate the influence of Mg 2+ ions on the efficiency of in vitro DNA repair.Therefore, we performed a photochemical DNA-repair assay with UV-induced (6-4)PP within ssDNA.ssDNA, oligo(dT 18 ) with two (6-4)PP lesions per molecule, was added in a 4:1 excess over fully reduced Vc(6-4), and the samples were illuminated with blue light at 445 nm, with an intensity of 5.9 mW•cm −2 (224 μmol•m −2 •s −1 ).Repair assays were carried out with a total blue-light illumination time of 60 min.DNA-repair was monitored via the absorption decrease at 325 nm.The absorption at 325 nm is significant for (6-4)PP lesions within DNA strands (52).For detection of a Mg 2+ effect, further measurements were done with additional 6 mM MgCl 2 .The repair activity of Vc(6-4) was quantified after a total illumination time of 60 min, with the absorption difference ΔA325nm and the amount of repaired (6-4)lesion, which were estimated with the initial concentration of oligo(dT 18 ) and the calculated, average amount of UV-induced (6-4)PP lesion.The results for the in vitro DNArepair with and without additional Mg 2+ ions are summarized in Fig. 5a.
Mg 2+ ions clearly enhance the in vitro DNA-repair activity of Vc(6-4) for ssDNA as the substrate.Without Mg 2+ ions, only ~10% of (6-4)PP lesion were repaired by the enzyme.The efficiency is fourfold higher (~40%) when Mg 2+ is present, but not as quantitative as observed before for the repair of a homogenous substrate by Ma et al. (60) or of the same oligo(dT 18 ) substrate using a eukaryotic-type (64) photolyase such as CraCRY (12).oligo(dT 18 ) strand was illuminated with UV-light to generate (6-4)PP, which appears randomly along the oligonucleotide strand.It is feasible that some fraction of UV-light induced (6-4)PP lesions is only poorly recognized as a substrate by Vc(6-4) due to the position in the oligo(dT 18 ) substrate.Nevertheless, the overall improved DNA-repair efficiency by Mg 2+ for Vc(6-4) is comparable to previous results for RsCryB and PhrB.Ma et al. (60) reported a poor repair efficiency in the absence of Mg 2+ for both prokaryotic (6-4) photolyases (~3%).However, the effect of Mg 2+ ions on repair efficiency is higher pronounced for RsCryB and PhrB with repair yields of 48% for PhrB and 82% for RsCryB after only 3 min of blue light irradiation.Overall, Vc (6-4) depends on Mg 2+ ions like the other prokaryotic (6-4) photolyases.Different repair yields are derived from experimental conditions, as we performed a photometrical assay with an inhomogeneously damaged oligo(dT)18 strand, whereas Ma et al. performed HPLC-based repair assays using a defined octamer nucleotide with a central (6-4)PP.  .Analysis of Vc(6-4) in regards to highly conserved regions, using ConSurf Webserver.The sequence is colored by conservation grade (see right lower panel for colored scale) from green (highly variable) to purple (highly conserved).The two highest conservation shells of the ConSurf analysis are shown in magenta (scale point 9) and in pink (scale point 8).The analysis is based on the structure of Vc(6-4) with the default setup of the ConSurf Server, with 500 selected homolog sequences as input data for the multiple sequence alignment, indicating a highly conserved core region of the catalytic domain as well as for the environments of the additional chromophore DLZ and the [4Fe-4S] cluster.
The binding of divalent ions takes place via two conserved aspartic acids (D180 and D256), which are also present in previous solved structures (Fig. 5b).The binding site is located closely to the putative DNA-binding groove.While D180 is located in a short α-helix a 9 within the interdomain loop, D256 is located in a loop between α 13 and α 14 within the catalytic domain.A WebLogo analysis of more than 11 000 sequences of prokaryotic (6-4) photolyases indicates a high degree of conservation for those residues (Fig. 5c).The binding site was previously determined via mutagenesis studies for PhrB.By exchanging the negatively charged aspartic acids to neutrally charged asparagines, both mutants PhrB_D179N and PhrB_D254N lack the typical enhancement of DNA-repair efficiency in the presence of divalent cations (7).The Mg 2+ effect is exclusively occurring within prokaryotic (6-4) photolyases and was excluded for other classes of photolyases like class I CPD photolyases and eukaryotic (6-4) photolyases (60).
Within this study, we present the high-resolution structure of the prokaryotic (6-4) photolyase from V. cholerae.Vc  possess the typical chromophore composition of prokaryotic (6-4) photolyases, including FAD as catalytic chromophore, the antenna DLZ, and the [4Fe-4S] cluster.Furthermore, Vc(6-4) shares prototypical behavior in regard to the lighttriggered reaction of those photolyases.Within the photoreduction, FAD will be reduced to the catalytic active species FADH − and in photoreactivation, the presence of Mg2 + enhances the enzymatic repair activity of Vc(6-4) drastically.However, despite this novel structural information, the exact mechanism of substrate recognition remains unclear.As mentioned before, a superimposition of Vc(6-4) with an eukaryotic (6-4) photolyase bound to dsDNA with a (6-4) lesion leads to several steric clashes, especially at the [4Fe-4S] clustering harboring roof-like subdomain.While the general binding motif, i.e. the lesion flipped out toward the active center, is sterically feasible, the arrangement of the corresponding double-stranded DNA strand remains elusive, at least for the 3 0 arm of the duplex DNA.

Figure 1 .
Figure 1.Photoreduction of Vc(6-4).(a) In vitro photoreduction of Vc(6-4) with 25 mM DTT and blue-light illumination.The labeled peaks a, b, c, and d within the T 0 -sample (0 min, black solid line, UV-Vis spectrum after purification) correspond to major absorption peaks in the blue-light region, namely, at 382, 420, 440, and 475 nm.Photoreduction experiments were carried out with blue light with a maximum emission wavelength of 455 nm (ILS-Intelligent LED Solutions) and a light intensity of 5.9 mW•cm −2 (224 μmol•m −2 •s −1 ), and several time points were measured in a range from 0 min up to 75 min illumination time.Arrows within the spectrum indicate time-resolved increases or decreases of absorption in different absorption ranges.The insets show an expanded scale in the absorption spectra from 500-800 nm.(b) The corresponding light minus dark difference spectra for different time points during photoreduction.

Figure 2 .
Figure 2. X-ray crystal structure and electrostatic surface analysis of Vc(6-4).(a) Domain-architecture of Vc(6-4) with the N-terminal α/β-domain (blue), the long linker region (light-orange), which is wrapped around the overall protein fold, and the catalytic all-α-helical domain (red).The antenna chromophore DLZ (stick representation, purple) is bound within the N-terminal domain and the catalytic active FAD (sticks representation, yellow) within the all-α-helical domain.The binding site for the iron-sulfur cluster [4Fe-4S] is part of the roof-like subdomain, an extension of the C-terminal domain.Closest edge-to-edge distances between the chromophores are shown in the lower panel.(b) Analysis of electrostatic surface potentials as calculated by APBS software.The putative DNA-binding site of Vc(6-4) shares the typical positive charged groove around a hole, which enables direct contact for DNA lesions toward the adenine moiety of the catalytic essential FAD chromophore.

Figure 3 .
Figure 3. Details of the chromophore binding sites of Vc(6-4).(a) Binding of FAD in the U-shaped conformation within the catalytic center.A water molecule (W33) forms a hydrogen bond with the N5 atom of the isoalloxazine-ring system.Further potential residues for interactions via hydrogen bonds are highlighted, and corresponding atom-atom distances are given in Angström ( Å).(b) The DLZ binding site, located at the N-terminal α/β domain with several nearby residues, involved in hydrogen bond networks between the chromophore and the protein.(c) The binding site for the [4Fe-4S] cluster is located in an C-terminal extended roof-like subdomain and is made up of four cysteines, which are highly conserved subgroup of prokaryotic (6-4) photolyases.

Figure 4 .
Figure 4. ConSurf analysis of Vc.Analysis of Vc in regards to highly conserved regions, using ConSurf Webserver.The sequence is colored by conservation grade (see right lower panel for colored scale) from green (highly variable) to purple (highly conserved).The two highest conservation shells of the ConSurf analysis are shown in magenta (scale point 9) and in pink (scale point 8).The analysis is based on the structure of Vc(6-4) with the default setup of the ConSurf Server, with 500 selected homolog sequences as input data for the multiple sequence alignment, indicating a highly conserved core region of the catalytic domain as well as for the environments of the additional chromophore DLZ and the [4Fe-4S] cluster.

Figure 5 .
Figure 5. Repair of (6-4)PP in DNA by Vc(6-4).(a) In vitro DNA-repair assay of Vc(6-4) for (6-4)PP and the influence of Mg 2+ -ions on overall DNArepair efficiency.The amount of repaired (6-4)-lesions within ssDNA are given in percentage for in vitro DNA repair without Mg 2+ (black dots) and with addition of MgCl 2 (red dots).Experiments were carried out with 5 μM fully reduced Vc(6-4), 20 μM oligo (dT)18 with an average of two (6-4)PP lesions per strand and 5 μM DTT.To investigate the influence of divalent cations, the identical condition with additional 6 mM MgCl 2 was used.The repair assays were carried out with blue light illumination at a maximum emission wavelength of 455 nm (ILS-Intelligent LED Solutions) and a light intensity of 5.9 mW•cm −2 (224 μmol•m −2 •s −1 ), and several time points were measured in a range from 0 min to 60 min illumination time.The measurements were carried out in triplicate, and the standard deviations for each time point are marked with error bars.(b) Structural alignment of both aspartic acids, which are responsible for coordination of Mg 2+ within the prokaryotic (6-4) photolyase.The residues of PhrB (D179, D254, colored in red), RsCryB (D175, D250, colored in green) and Vc(6-4) (D180, D256, colored in light-blue) are shown.The position of the Mg 2+ ion was excerpted from the RsCryB structure, which was crystalized in the presence of those ions.(c) WebLogo analysis based on an alignment of all entries of PTHR38657 (containing >11 000 sequences of prokaryotic (6-4) photolyases and cryptochromes.The region of the interdomain-loop region including D180 and an excerpt of the α-helical domain with the highly conserved D256 are shown.

Table 1 .
Data collection and refinement statistics.