Mitrocomin from the Jellyfish Mitrocoma Cellularia with Deleted C

Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Mitrocomin from the jellyﬁsh Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

Ludmila P. Burakova a,1, Pavel V. Natashin a,b,1, Svetlana V. Markova a, Elena V. Eremeeva a, Natalia P. Malikova a, Chongyun Cheng b, Zhi-Jie Liu b,c,d,⁎, Eugene S. Vysotski a,⁎ a Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia b National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China c Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China d iHuman Institute, ShanghaiTech University, Shanghai 201210, China article info abstract

Article history: The full-length cDNA genes encoding five new isoforms of Ca2+-regulated photoprotein mitrocomin from a small Received 8 April 2016 tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Received in revised form 30 June 2016 Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and sub- Accepted 30 June 2016 sequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed sugges- Available online 2 July 2016 tion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2+-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2+-discharged aequorin, but different from Ca2+-discharged obelins and clytin which fluorescence is red-shifted by 25– 30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specificactivityandefficiency of apo- mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties. © 2016 Elsevier B.V. All rights reserved.

1. Introduction molecular oxygen or any other cofactor, the reaction strikingly differs from that of classical bioluminescent systems in which an enzyme Ca2+-regulated photoproteins are “precharged” bioluminescent (luciferase) catalyzes the oxidation of a smaller organic substrate mole- proteins that are triggered to emit light by addition of Ca2+ or certain cule (luciferin) with the creation of an excited state and light emission. other inorganic ions [1]. The reaction does not require the presence of This feature prompted Shimomura and Johnson to coin the term molecular oxygen or any other cofactor – the photoprotein and the trig- “photoprotein” to describe proteins that serve as sole organic molecular gering ion are the only components required for light emission. Since species in bioluminescent reaction systems [2]. the energy emitted as light is derived from the “charged” photoprotein, Though other kinds of photoproteins have been described, the ma- the molecule can react only once, i.e. it does not “turn over” as an en- jority of the presently known photoproteins are stimulated to lumines- zyme does. In this respect, as well as in the lack of a requirement for cence by calcium. Ca2+-regulated photoproteins are responsible for bioluminescence of a variety of marine organisms [1]. The best known and studied of these is aequorin, ﬁrst isolated in 1962 by Shimomura ⁎ Corresponding authors. et al. from the jellyﬁsh Aequorea victoria [3].AllCa2+-regulated E-mail addresses: [email protected] (Z.-J. Liu), [email protected] (E.S. Vysotski). photoproteins isolated to date are one-subunit globular proteins, in 1 These authors contributed equally to this work. the inner cavity of which there is a non-covalently bound peroxy-

http://dx.doi.org/10.1016/j.jphotobiol.2016.06.054 1011-1344/© 2016 Elsevier B.V. All rights reserved. L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 287 substituted coelenterazine molecule, 2-hydroperoxycoelenterazine [4– and the choice of which one to use depends on the biological task 6]. The bioluminescence reaction is an oxidative decarboxylation of 2- to be addressed. hydroperoxycoelenterazine with the elimination of one mole of carbon The amino acid sequences of hydromedusan Ca2+-regulated dioxide and the generation of the protein-bound product (called photoproteins from different organisms reveal a high degree of identity coelenteramide) in an excited state [7,8]. The excited coelenteramide (76–64%) [42,43]. These photoproteins, except mitrocomin, contain Pro then relaxes to its ground state with the production of blue light of a residue at C-terminus. The C-terminal Pro is important for photoprotein broad spectrum with its maximum in a range 465–495 nm, depending bioluminescence because its deletion or replacement destroys the lumi- on the photoprotein type [9]. nescence capacity of aequorin [44,45]. However, it was found later that Although Ca2+-regulated photoproteins have been detected in the addition of a peptide consisting of eight amino acid residues to the many (N25) different coelenterates [10], cloning and sequence analysis C-terminus of a cysteine-free aequorin mutant [46] or Tyr residue to have been performed only for four hydromedusan photoproteins the obelin C-terminus [47] has no drastic effect on their biolumines- (aequorins from Aequorea victoria [11–13], Aequorea coerulescens [14], cence. In contrast to other hydromedusan photoproteins, a C-terminal and Aequorea macrodactyla [15], clytins from Clytia gregaria [16–18] residue in mitrocomin is tyrosine. The reason of that is unclear. and Clytia hemisphaerica [19], mitrocomin from Mitrocoma cellularia In the present study, we report the cloning of cDNA genes encoding [20], obelins from Obelia longissima [21,22] and Obelia geniculata [23]) several isotypes of the Ca2+-regulated photoprotein mitrocomin from and three photoproteins of ctenophores [24–28], which are extremely the jellyfish Mitrocoma cellularia, their expression in E. coli cells, purifi- sensitive to light, i.e. in contrast to hydromedusan photoproteins they cation and characterization of several mitrocomin isospecies, and the lose the ability to bioluminescence on exposure to light over the entire crystal structure of mitrocomin determined at 1.30 Å resolution. In addi- absorption spectrum [1]. Apophotoproteins expressed in Escherichia coli tion, we quantitatively characterize an effect of deletion of the C-termi- can be converted into active photoproteins by incubating them with nal tyrosine on mitrocomin bioluminescence. 2+ coelenterazine under Ca -free conditions in the presence of O2 and re- ducing agents [29]. While coelenterazine binding with apoprotein re- 2. Materials and Methods quires milliseconds [30], its following conversion into 2-hydroperoxy adduct occurs much slower [31]. 2.1. Collection of the Jellyfish M. cellularia and Preparation of the cDNA Ex- Ca2+-regulated photoproteins draw a strong interest due to their pression Library wide analytical potential. Their main application derives from the ability to emit light upon Ca2+ binding. Natural aequorin and obelin embedded The jellyfish M. cellularia was collected in August 2001 at Friday Har- in living cells were the first indicators of intracellular calcium and have bor Laboratories, University of Washington. The tissue sample been used to detect calcium ions in various types of living cells [32,33]. (~100 mg) was excised from the outer bell margin containing Although Ca2+-regulated photoproteins, mostly aequorin, are still the photocytes of only one jellyfish specimen and immediately used for indicators of choice for some applications, they have now been largely mRNA isolation. The poly(A) + mRNA was directly isolated with eclipsed by the fluorescent tetracarboxylate calcium indicators intro- Straight A's mRNA Isolation Kit (Novagen) and approximately 1 μg duced by Tsien [34]. These substances rapidly gained popularity because (~1/10 part) was employed to synthesize cDNA. The synthesis of cDNA of their ready availability, stability, and ease of introduction into cells and the cDNA library construction were carried out using the SMART [35]. The cloning of cDNAs encoding apophotoproteins opened new av- cDNA library construction kit (Clontech) according to the accompanying enues for utilizing photoproteins, by expressing the recombinant protocol. The first-strand cDNA was synthesized with PowerScript re- apophotoprotein intracellularly, then adding coelenterazine externally verse transcriptase (Clontech). Then the single strand cDNA was ampli- which diffuses into the cell and forms an active photoprotein. Such fied by LD PCR with Advantage cDNA PCR kit (Clontech) during 18 cells have, in effect, a “built-in” calcium indicator with many advantages thermal cycles using the following cycling parameters: 95 °C for 30 s, over fluorescent probes. Ca2+-regulated photoprotein, since it is a 68 °C for 6 min, followed by 17 cycles (95 °C for 10 s, 68 °C for 6 min). protein, can be engineered to induce specific targeting sequences, These procedures were carried out at Friday Harbor Laboratories. permitting selective localization of the photoprotein to a cell region The synthesized cDNA was digested with Sfi I, size-fractionized, and of interest; the background in photoprotein measurements is very cloned into Sfi I site of the pTriplEx2 vector (Clontech) expressing cDNA low, resulting in a high signal-to-noise ratio, wide dynamic range, inserts in all three reading frames. The resulting unamplified plasmid and low Ca2+-buffering effect [36–38]. Nowadays this approach is cDNA library in E. coli strain XL1-Blue contained approximately widely applied. The major drawback of photoproteins is the low 2.2 × 105 independent recombinant clones. light signals from cells because the photoprotein does not “turn over” as an enzyme does. It is not a hindrance when recordings 2.2. Library Screening for Mitrocomin from microplate wells with cells expressing photoprotein are performed. However, it is often required to image Ca2+ transients at a The clones containing cDNA mitrocomin isoforms were isolated single cell level. Although such experiments with aequorin have from cDNA expression library with functional screening which we suc- been performed [39,40], they are technically complicated requiring cessfully used to isolate the cDNA genes encoding other bioluminescent the special imaging systems. Recently, genetically encoded calcium and fluorescent proteins [48]. For screening, the unamplified expression indicators based on fluorescent proteins have been developed. The plasmid cDNA library was plated at low density of ~1500 recombinant E. best known of these are cameleons, camgaroos, and pericams [36, coli colonies per Petri dish. An imprint of the colony pattern was obtain- 41]. Although they have some advantages over chemical fluorescent ed by applying a dry nitrocellulose filter to the primary plate. The replica probes (no dependence of sensitivity and Ca2+-binding kinetics on filter was then turned over, applied to a fresh sterile agar plate, and left indicator concentration, precise expression in targeted intracellular on the plate for the bacteria to grow. The replica filters were grown until compartments) and photoproteins (ratiometric Ca2+ concentration the clearly visible bacterial colonies appeared. Then the bacteria were measurements, reversible Ca2+-dependent fluorescent response, scraped off the filter with a sterile spreader into LB medium supple- no need for specific cofactors, relatively bright fluorescence, and mented with ampicillin (50 μg/mL). The suspension was poured into a comparatively high temporal resolution), these Ca2+ indicators culture tube, and gene expression was induced with 1 mM IPTG for based on fluorescent proteins also have some drawbacks: small dy- 2 h at 37 °C. The cell pellet from 2 mL of suspension was sonicated in namic range of fluorescence intensity as compared with that of fluo- 0.3 mL of 5 mM EDTA, 20 mM Tris-HCl pH 7.2 at 0 °C. The crude cell ly- rescent dyes and sensitivity of fluorescence to pH [36,41].Thus,all sate was charged with coelenterazine (Prolume, Pinetop, USA) (final Ca2+ indicators have their intrinsic advantages and shortcomings, concentration 10−7 M) in the presence of 10 mM DTT and assayed for 288 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 bioluminescence activity. The next replica from a positive primary plate 2.5. Bioluminescent Assay was cut into sectors for more detailed location of positive colonies and was analyzed as above. Then the individual colonies from the positive During screening, bioluminescence was measured by rapid injection areas were isolated and assayed for bioluminescence activity after of 0.2 mL of 0.1 M CaCl2 in 0.1 M Tris-HCl pH 8.8 into a luminometer cell IPTG induction. containing a crude cell lysate in 0.5 mL of the assay buffer 10 mM EDTA, The nucleotide sequences for mitrocomin cDNA genes reported in 0.1 M Tris-HCl pH 8.8. The bioluminescence of pure photoproteins was this paper have been submitted to the GenBankTM/EBI Data Bank measured by rapid injection of 10 μL photoprotein solution into a with the accession numbers: MC1 (KF882008), MC2 (KF882009), MC3 luminometer cell containing 490 μL of 2 mM CaCl2 in 50 mM Tris-HCl (KF882010), MC4 (KF882011), MC5 (KF882012), MC6 (KF882013), pH 8.8. All bioluminescence measurements were carried out at room MC7 (KF882014), and MC8 (KF882015). The nucleotide sequence for temperature. another isoform of obelin cDNA gene from O. geniculata has been submitted to the GenBankTM/EBI Data Bank with the accession number OG2 (KF882016). 2.6. Spectral Measurements

Spectral measurements were carried out with a Varian Cary Eclipse 2.3. Expression of the Recombinant Photoproteins in E. coli and Purification spectrofluorimeter (Agilent Technologies). Emission spectra were corrected using the computer program supplied with the instrument. The constructs for direct expression of apo-mitrocomin isoforms Bioluminescence spectra were measured in a buffer 50 mM Bis-Tris- were produced by amplification of the coding regions only from original propane pH 7.0. Bioluminescence was initiated by the injection of pTriplEx2-MC(#) with specific primers containing NdeI (forward prim- CaCl solution in the same buffer. The concentration of free calcium er) and XhoI (reverse primer) sites for the following subcloning into 2 was ~0.5 μM to provide an approximately constant light level during NdeI⁄XhoI sites of the expression vector pET22b + (Novagen). The the spectral scan. If a substantial change in bioluminescence intensity resulting plasmids were named pET22-MC(#) with a corresponding took place during the spectral scan, the data points were also corrected number of cDNA gene. After verification of the nucleotide sequences, for bioluminescence decay. Fluorescence and excitation spectra were the obtained expression plasmids were introduced into E. coli strain measured immediately after disappearance of a bioluminescent signal. BL21(DE3)-CodonPlus-RIPL (Stratagene). For production of the recom- All spectral measurements were carried out at room temperature. binant apo-mitrocomin isoforms, E. coli cells transformed with the corresponding expression plasmids pET22b-MC(#) were cultivated at 37 ° C and induced with 1 mM IPTG when the culture reached an OD of 600 nm 2.7. Apophotoprotein Preparation, Fluorescence Measurements, and Deter- 0.8. After addition of IPTG, the cultivation was continued for 3 h. mination of Apparent K of the Apophotoprotein-Coelenterazine Complex The E. coli cells were harvested by centrifugation (3500 g for 10 min D at 4 °C), the pellet was resuspended in 20 mM Tris-HCl pH 7.2 (1:5, w/ The apophotoproteins for determination of apparent dissociation v), and disrupted by sonication (20 s × 6) at 0 °C. The recombinant apo- constant (K ) of the apophotoprotein-coelenterazine complex were ob- mitrocomin was purified from inclusion bodies and charged with D tained according to the procedure described elsewhere [51]. The Trp coelenterazine as previously reported for the recombinant obelin [23, fluorescence was measured with a Varian Cary Eclipse spectrofluorime- 49,50]. The apo-mitrocomin obtained after extraction with 6 M urea ter (Agilent Technologies) in 5 mM EDTA, 10 mM DTT, 20 mM Tris-HCl and purification on a DEAE-Sepharose Fast Flow column was concen- pH 7.2 at 20 °C. Excitation was at 295 nm (slit 5 nm). The fluorescence trated on Amicon centrifugal filters (Millipore). To fold apo-mitrocomin, emission spectra were corrected with the computer program supplied the concentrated sample containing 6 M urea was diluted 20-fold at 4 °C with the instrument. All the spectra were taken using a standard quartz with a freshly prepared solution containing coelenterazine (~1.1 mol cuvette (1 × 1 cm) in a 1-mL initial volume with varied coelenterazine per mole of apophotoprotein) in 5 mM EDTA, 10 mM DTT, 20 mM additions in 5- to 10-μL portions up to saturation. To assess fluorescence Tris-HCl pH 7.2. Then, a mixture was incubated overnight at 4 °C to con- quenching, only the changes of fluorescence intensity at 336 nm were vert apo-mitrocomin into an active photoprotein. The coelenterazine used. The fluorescence intensities were corrected for dilution due to concentration in methanol stock solution was determined spectropho- the addition of coelenterazine, for methanol influence on Trp fluores- tometrically using the molar absorption coefficient ε = 435 nm cence, scattered light, and for the inner filter effects of a protein and 9800 M−1 cm−1 in methanol [1]. To separate apoprotein from the added coelenterazine. To evaluate the inner filter effects, absorbance charged mitrocomin, the samples were additionally purified by ion-ex- measurements were performed at excitation and emission wave- change chromatography on Mono Q column (GE Healthcare) equilibrat- lengths, and fluorescence (F) was corrected using the equation: ed with 5 mM EDTA, 20 mM Tris-HCl pH 7.2. The charged mitrocomin was eluted with a linear salt gradient (0–0.5 M NaCl in 5 mM EDTA,

þ 20 mM Tris-HCl pH 7.2) (Fig. S1). The mitrocomin isospecies and A295 A336 F ¼ F e 2 ð1Þ mitrocomin mutant with deleted C-terminal Tyr were of a high purity unc according to SDS-PAGE. The protein concentration was determined with both the D c where A and A are absorbance of a protein and ligand at excitation Bio\\Rad protein assay kit and the use of absorption for aequorin at 295 336 and emission wavelengths, respectively, and F is the uncorrected 460 nm (A = 0.81) [1]. unc 1%, 1cm, 460 nm fluorescence. The apparent dissociation constant of the apophotoprotein- 2.4. Site-Directed Mutagenesis coelenterazine complex was determined using the quenching of apophotoprotein Trp fluorescence upon binding to coelenterazine. The Mitrocomin mutant with deletion of C-terminal tyrosine (MC2dY) analysis assumes that the fraction of a bound ligand is equal to the was constructed using the QuikChange site-directed mutagenesis kit ratio of the fluorescence quenching (Q = Fo − Fq)tothemaximum (Stratagene). Forward and reverse primers were designed according quenching (Qmax = Fo − Fqmax), where Fo, Fq,andFqmax are fluorescence to the manufacturer's protocol. The pET22b-MC2 expression plasmid intensity at 336 nm measured in the absence of added ligand, the was used as a template for mutagenesis. The presence of the mutation quenched fluorescence intensity in the presence of ligand, and the max- was verified by sequencing. The mitrocomin mutant was purified as de- imum fluorescence quenching at a saturating level of ligand. The appar- scribed above for mitrocomin isoforms. ent dissociation constants were calculated by fitting the relative L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 289

fluorescence emission to Equation 2 [30]: Table 1 Summary of crystallographic statistics. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðÞþ þ − ðÞþ þ 2− Crystal name Mitrocomin Q C L KD C L KD 4CL ¼ ð2Þ Q 2 Data processing max Resolution range (Å) 50.00–1.30 (1.35–1.30) Wavelength (Å) 0.9795 Space group P2 where C, L, and KD are apophotoprotein and coelenterazine concentra- 1 tions, and apparent dissociation constant, respectively. The concentra- Cell dimensions (Å) a = 48.90 b = 69.68 c = 49.93 Unit cell angles (°) α = 90.00 β = 90.03 γ = 90.00 tions of apo-mitrocomin and its mutant for these measurements were Unique reflections 54,457 (234) determined using the corresponding molar extinction coefficients at Completeness (%) 66.42 (2.83) 280 nm calculated with the ProtParam tool (http://us.expasy.org/ Mean I/σ (I) 17.65 (1.67) tools/protparam-doc.html). Rmerge (%) 6.3 (26.4) Multiplicity 3.3 (1.2)

2.8. Ca2+ Concentration-Effect Curves Refinement Resolution range (Å) 40.59–1.30 Reflections used (free) 54,441 (2008) Mitrocomin isotype MC2 and its mutant with deletion of C-terminal R (R ) 16.13% (19.04%) 2+ work free Tyr for determination of Ca concentration-effect curves were pre- No. of protein atoms 3246 pared in accord with the procedure described elsewhere in details [52, No. of ligand atoms 68 53]. The Ca-EGTA buffers (total [EGTA] = 2 mM) and simple dilutions No. of solvent atoms 469 Mean B (Å2) 19.20 of CaCl2 (in a Chelex-scrubbed solution of 150 mM KCl, 5 mM PIPES, 2+ μ RMSD bond lengths (Å) 0.013 pH 7.0) were used to establish Ca concentrations below 10 M and RMSD bond angles (°) 1.41 for higher Ca2+ concentrations, respectively [54]. A 10-μl aliquot of Values in parentheses are for the highest resolution shell. photoprotein sample with a concentration of 1–5 μM was injected by a constant rate syringe CR 700–20 (Hamilton, USA) into 1 mL test solution. Light intensity (L) measurements were converted to units of L/Lint COOT [59].Therefined model of mitrocomin was deposited at the Pro- by first calculating L/Lmax and then multiplying L/Lmax by the maximum tein Data Bank (PDB) with the access code 4NQG. Visualization and su- peak-to-integral ratio (Lmax/Lint), determined from kinetic measure- perposition of the molecular structures were performed using the ments carried out under the same conditions with the same PyMOL program (Delano ScientificLLC). photoprotein sample. All the measurements were performed at 20 °C using a luminometer equipped with a temperature-stabilized cuvette 3. Results block and neutral-density filters with transmission coefficients to match the intensity of the light signals, which typically vary about 8 3.1. cDNA and Amino Acid Sequences log units from low to saturated calcium concentrations. 2+ The apparent dissociation constant (Kd) was estimated from Ca The obtained M. cellularia cDNA library yielded approximately 5 concentration-effect curves using a two-state model [55].TheKd values 2.2 × 10 independent recombinant bacterial colonies with N90% were calculated as the mean of the apparent dissociation constants recombinants. After functional screening of about 30,000 colonies of determined from 3 Ca2+ concentration-effect curves for each cDNA library, the eight independent positive clones revealing photoprotein. The stated error is the standard deviation. photoprotein activity were isolated. The isolated full-length cDNA genes were very similar, with some nucleotide differences including dif- 2.9. Crystallography ferent length varying from 887 to 900 bp excluding poly(A) tail (Fig. S2). Each cDNA gene includes the same 81 bp 5′-untranslated region up- For crystallization, mitrocomin MC2 obtained after ion-exchange stream of the first start codon and different 3′-untranslated regions end- chromatography was exchanged into a buffer containing 2 mM EDTA, ing in different places after the TAA stop codon and varying from 209 to 10 mM Bis-Tris pH 6.5 and concentrated to 12.5 mg/mL using Millipore 222 bp fragments followed by a terminal poly(A). All isolated cDNA centrifuge tubes. For these experiments, mitrocomin charged with a genes contain an open reading frame of 594 bp, which encodes 198 high purity coelenterazine (JNC Corporation, Japan) has been used. amino acid polypeptides revealing a high degree of identities of ~97– The Mosquito crystallization robot (TTP LabTech, UK) and commercially 98% (Fig. S3) with the MI17 mitrocomin (P39047) cloned before [20]. available screening kits (Emerald BioSystems, Hampton Research) were The eight isolated cDNA genes encoding mitrocomin distinctly di- used for screening initial crystallization conditions. Optimization was vided into two groups, 64R and 64Q isotypes, based on the presence performed manually with the hanging-drop vapor-diffusion technique of specific patterns of nucleotide substitutions (Fig. S2). The MC1, by mixing of 2 μLproteinand2μL precipitant solutions. The best condi- MC6, and MC7 cDNA genes encoding the same protein, denoted as tion for mitrocomin crystallization was 1.4 M sodium citrate tribasic MC1 mitrocomin 64Q-isotype, have the common 689A and 272A nucle- dihydrate solution, 0.1 M HEPES pH 7.0 (Index Kit, Hampton Research). otide substitutions in comparison with other isolated cDNA genes. The The mitrocomin grew as a big light yellow cubic shape crystal after other five isolated cDNA genes (MC2, MC3, MC4, MC5, and MC8) have 10 days at 4 °C. For X-ray diffraction analysis, the crystal was picked been attributed to 64R-isotype group because in the same positions up directly from the crystallization drop using a fiber loop, and then they contain 689T and 272G nucleotide substitutions leading to appear- flash-cooled in liquid nitrogen. ance of 64R in mitrocomin sequence (Fig. S2). Among these cDNAs, MC3 Data were collected at Beamline 22-ID (Advanced Photon Source at and MC8 have almost identical consensus sequences excluding the lack Argonne National Laboratory, IL, USA). Diffraction data were indexed, of the last 5 bp in 3′-noncoding sequence of MC8. Thus, the MC3 and integrated and scaled (Table 1) using the HKL2000 software suite [56]. MC8 cDNAs encode MC3 mitrocomin 64R-isotype differing in one The phases were determined by molecular replacement with PHASER amino acid from MC1 mitrocomin 64Q-isotype. The MC2, MC4, and [57] using the structure of aequorin (PDB code 1EJ3) because aequorin MC5 cDNA genes of 64R group have additional nucleotide differences displays the highest degree of identity with mitrocomin among other as compared to MC3/MC8 consensus sequence resulting in some hydromedusan photoproteins. There are two mitrocomin molecules amino acid replacements (Fig. 1). (A and B) in one asymmetric unit. The final models were refined with Mitrocomin amino acid sequence reveals the highest and the lowest PHENIX [58]. Manual adjustments to the model were performed using degree of identity with that of aequorin and clytin, respectively; 290 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297

Fig. 1. Scheme of the origin of apparent diversity of isotypes for photoprotein mitrocomin deriving from two allelic genes of one specimen genome of M. cellularia. mitrocomin MC3 shows 70.3% of sequence identity with aequorin AEQ1 highly compact and globular spatial structure conﬁgured by eight α-he- whereas with clytin CLI the identity is only 59.8% (Fig. 2). However, the lices assembled into two cup-shaped N-terminal and C-terminal do- main distinction of mitrocomin amino acid sequence from those of mains. These domains form a globular structure with internal cavity in other hydromedusan photoproteins is yet an additional Tyr residue at which 2-hydroperoxycoelenterazine molecule resides. The root-mean- C-terminus. square deviation (RMSD) of the main chain atoms of mitrocomin versus aequorin is 0.50 Å, mitrocomin vs. obelin – 0.50 Å, and mitrocomin vs. 3.2. 3.2. Crystal Structure clytin – 0.66 Å (Fig. 3B). The substrate-binding pocket is highly hydrophobic and is Although mitrocomin itself is a monomer just as aequorin [4] it was formed by the residues originating from each of the α-helices of crystallized as a dimer (Fig. 3A). There are almost no differences be- mitrocomin molecule. In addition, several hydrophilic side chains tween two molecules except some side chains of the surface residues of His24, His177, Tyr140, and Tyr192 are also directed internally. which reveal slightly different orientation. Similar to other Ca2+-regu- All of these residues form hydrogen bonds with different atoms of lated photoproteins [4–6], the mitrocomin molecule has the same 2-hydroperoxycoelenterazine molecule (Fig. 4)andtheyare

Fig. 2. Comparison of mitrocomin MC3 amino acid sequence with those of isotypes of other Ca2+-regulated photoproteins: mitrocomin MI17 (P39047) [20] from M. cellularia, aequorins AEQ1(M16103) [13] and A440 (AQ440, L29571) [12] from A. victoria, obelins OG1 (AAL86372) [23] and OG2 (KF88201) from O. geniculata, and clytins CLI (CAA49754) [16] and CL3 (GU721042) [18] from C. gregaria. Two isotypes with maximal sequence differences of each species were compared. Letters colored by red show identical, blue letters represent similar and black letters show nonidentical residues. Amino acid replacements, making new MC3 mitrocomin isotype different from MI17 isotype cloned before are indicated by cyan boxes. Ca2+-binding sites I, II, and III are marked with yellow. The residues forming the substrate binding pocket are shown by grey boxes. GenBank accession numbers for sequences are given in parentheses. L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 291

Fig. 3. Overall crystal structure of the mitrocomin MC2 (A) and stereoview of the superimposition of different photoproteins (B): mitrocomin (blue), aequorin (green), obelin (red), clytin (yellow). The 2-hydroperoxycoelenterazine molecules are displayed by the stick models in the center of a protein and are colored according to the structure colour. Mitrocomin was obtained with two molecules (A and B) per asymmetric unit. The comparison has been performed for molecule A.

conserved for all Ca2+-regulated photoproteins (Fig. 1). The elec- situated. This condition apparently optimizes the efficient population tron-density map of 2-hydroperoxycoelenterazine molecule clearly of the first electronic excited state of the product coelenteramide and fa- shows the presence of two oxygen atoms, consistent with peroxide vors a high quantum yield for its fluorescence. substitution in C2 position [7] (Fig. 4B). The hydroperoxide is stabi- Although mitrocomin has a Tyr residue at C-terminus, the hydrogen lized by a strong hydrogen bond (2.66 Å) between the second oxy- bond network is essentially identical (Fig. 5D) to that of other gen and the hydroxyl group of Tyr192. There are also two water photoproteins. Similar to obelin, aequorin, and clytin, the oxygen atom molecules in the internal cavity of mitrocomin which are found in of carboxylic group of C-terminal Tyr190 is hydrogen bonded with Nη the same positions as in aequorin [4], obelin [60], and clytin [6]. of Arg. However the second hydrogen bond with Nε of Arg in Thus, the overall crystal structure and the substrate-binding pocket mitrocomin is formed by carbonyl oxygen of the preceding Pro197 in- of mitrocomin display a high degree of similarity with the other struc- stead of the second oxygen of carboxylic group of Pro in other tures of hydromedusan photoproteins. photoproteins (Fig. 5). All hydromedusan photoproteins, except mitrocomin contain Pro residue at C-terminus (Fig. 1) [42,43]. This proline is important for 3.3. 3.3. Properties of Mitrocomin Isoforms photoprotein bioluminescence because its deletion or replacement destroys the luminescent capacity of aequorin [44,45]. The spatial struc- The bioluminescence properties have been studied for mitrocomins tures of photoproteins [4,60] lay a rational molecular basis for MC1, MC2, MC3, and MC5 (Fig. 1). A summary of the mitrocomin char- explaining Pro function. The C-terminal Pro supports an active acterization is given in Table 2, and a portion of the supporting data ap- photoprotein conformation through hydrogen bond interactions be- pears in Fig. 6A. tween its oxygen atoms and nitrogen atoms of Arg found in the N-ter- The specific bioluminescence activities of mitrocomin isoforms were minal α-helix (Fig. 5A, B, C). As a result the N-andC-terminal found to differ from each other. Even MC1 and MC3 mitrocomin domains form a closed conformation with solvent-inaccessible internal isotypes differing in only one amino acid residue (Q64R) show the cavity in the center of which the 2-hydroperoxycoelenterazine is 1.6-fold difference of specific activities. The mitrocomin MC2 bearing 292 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297

Fig. 4. Stereoviews of 2-hydroperoxycoelenterazine molecule with the key residues facing into the substrate-binding cavity of mitrocomin (A) and the electron-density map of peroxy- substituted coelenterazine bound within photoprotein internal cavity (B). Hydrogen bonds are shown as dots; water molecules are presented as red balls. additional mutation (Q109R) as compared to MC3, displays a biolumi- hydromedusan photoproteins, i.e. the charged photoproteins have nescence activity practically equal to that of MC3 isotype. In contrast, maxima at 280 and 460 nm and a shoulder at ~310 nm, and Ca2+- MC5 isotype with two additional mutations (E92G/Q176R) displays a discharged mitrocomins have no absorption in the visible range specific activity much lower than other mitrocomin isoforms. The biolu- but exhibit absorption at 345 nm (data not shown) [1,18,23].The minescence decay constants are also different but not so much com- maxima of bioluminescence spectra of mitrocomin isoforms are at pared to the specific activities. It should be noted that the decay λmax = 473–474 nm (Table 2, Fig. 6A) and practically coincide kinetics of all mitrocomin isotypes can be satisfactorily characterized with that of mitrocomin MC17 (λmax = 470 nm) cloned earlier by a single rate constant, i.e. similar to wild type aequorin but differing [20]. The small difference is rather a result of some inaccuracy of from wild type obelin which kinetics is described by a two-exponential bioluminescence spectrum determination than the distinction of decay function, and consequently by two rate constants [52]. The cause their spectral properties. The fluorescent spectrum of Ca2+- of difference in specific bioluminescence activities of mitrocomin iso- discharged mitrocomin is almost identical to the light emission forms is unclear. Although these isotypes differ in some amino acids, spectrum (Fig. 6A). these residues are not involved directly in the formation of a substrate-binding cavity and in bioluminesscence reaction. Nevertheless, 3.4. 3.4. Properties of Mitrocomin Mutant with Deleted C-Terminal Tyr these results are consistent with those obtained for natural isoforms of aequorin from A. victoria and obelin from O. longissima which specific To estimate the impact of C-terminal Tyr on mitrocomin properties, bioluminescent acivities and decay constants also differ [61,62]. the tyrosine was deleted by site-directed mutagenesis using the All mitrocomin isotypes and their Ca2+-discharged conforma- mitrocomin MC2 as a template. The properties of MC2dY mutant are tion states display the absorption spectra characteristic of other summarized in Table 3, Figs. 6B and 7. Noteworthy, in contrast to L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 293

Fig. 5. Hydrogen bond network formed by C-terminal amino acid and Arg located in N-terminal α-helix in obelin (A) (PDB code 1QV0), aequorin (B) (PDB code 1EJ3), clytin (C) (PDB code 3KPX), and mitrocomin (D). Hydrogen bonds are shown as dots. aequorin [44,45], the deletion of C-terminal residue increases spe- Fig. 7 shows the effect of C-terminal Tyr deletion on sensitivity of cific bioluminescence activity by a factor of 1.6, accompanied by mitrocomin to Ca2+.TheverticalrangeoftheCa2+ concentration-effect the2-folddecreaseofadecayconstantascomparedtomitrocomin curve for MC2dY mutant is slightly reduced as compared to that of wild MC2. Although C-terminal residue is far away from the substrate- type mitrocomin. It is due to both the increase of Ca2+-independent lu- binding cavity, the MC2dY mutant bioluminescence spectrum is minescence (Table 3) and the decrease of L/Lint ratio at saturating calci- 2+ shifted towards a shorter wavelength for 15 nm (λmax =458nm) um concentrations. The maximum slopes of the Ca concentration- compared to wild type mitrocomin (λmax = 473 nm). However, effect curves for wild type and MC2dY mitrocomins are 2.28 and 2.15, Ca2+-discharged MC2dY mutant retains fluorescence maximum respectively, pointing out that 3 Ca2+ are required for bioluminescence 2+ at λmax = 472 nm, i.e. at the same wavelength as Ca -discharged of wild type mitrocomin and MC2dY mutant. For quantitation of the in- mitrocomin MC2 (Table 2 and Fig. 6). It should be pointed out that fluence of deletion of C-terminal Tyr on Ca2+ sensitivity of mitrocomin, although light emission maximum of MC2dY is shifted as compared the apparent dissociation constant (Kd)wasused[55]. Although Tyr res- to wild type mitrocomin, the shapes of their bioluminescence spec- idue at C-terminus is far away from Ca2+-binding sites of mitrocomin, tra are exactly the same. its deletion reduces the affinity to calcium by a factor of 1.5 (Table 3).

Table 2 Bioluminescence properties of mitrocomin isoforms and mitrocomin mutant with deletion of C-terminal tyrosine.a

§ 10 a −1 Mitrocomin Speciﬁc bioluminescence activity, RLU/mg, ×10 Bioluminescence, λmax, nm Fluorescence, λmax,nm kdecay, s MC1 172 ± 1.0 473 473 0.92 ± 0.01 MC2 118 ± 1.0 473 472 0.90 ± 0.04 MC5 1.46 ± 0.03 474 473 1.06 ± 0.02 MC3 109 ± 2.0 473 473 1.10 ± 0.02

a Average values of three independent preparations of mitrocomin isotypes. 294 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297

Fig. 6. Normalized bioluminescence (solid line) and ﬂuorescence (dashed line) spectra of mitrocomin MC2 (A) and its mutant MC2dY with deletion of C-terminal Tyr (B).

The term “Ca2+-independent luminescence” denotes light emission groups according to the specific nucleotide patterns (Fig. S2). The con- of a photoprotein in the absence of calcium [55] caused by a spontane- sensus sequences for each group are MC1 and MC3 genes which encode ous decarboxylation reaction of 2-hydroperoxycoelenterazine initiated two mitrocomin isotypes, 64Q and 64R, respectively. The MC3-64R by structural fluctuations of a photoprotein molecule. The level of group also includes the cDNA genes encoding isotypes with additional Ca2+-independent luminescence varies for photoproteins from differ- mutations (Fig. 1 and Fig. S2) which are definitely derived from MC3- ent organisms and semisynthetic photoproteins charged by different 64R gene. The additional nucleotide substitutions in non-consensus se- coelenterazine analogs [52,53] and correlates well with a photoprotein quences of both groups amounting to 1–3 per cDNA gene (11 for all stability. Deletion of C-terminal Tyr increases Ca2+-independent lumi- cloned cDNAs) are randomly distributed through the isolated sequences nescence as compared to wild type mitrocomin (Table 3) and conse- and look as accidental mutations of products of only two original genes quently makes mutant photoprotein less stable. The gain of Ca2+- (Fig. S1). Taking into account that M. cellularia cDNA library was pre- independent luminescence might be caused by partial disturbance of pared from a part of a single animal, we might conclude that all isolated hydrogen bond network which brings together C-andN-terminal cDNA genes divided into 64R and 64Q groups are independent RNA- parts of mitrocomin molecule (Fig. 5D). transcripts of only two allelic genome genes situated in a single chromo- Among recombinant Ca2+-regulated photoproteins tested, only some locus of a heterozygous organism. These genome allelic genes cor- mitrocomin reveals very low bioluminescence intensity at its ex- respond to the MC1 and MC3/8 consensus cDNA sequences and encode pression in mammalian cells [52]. It was suggested that inefficient two variants of mitrocomin, MC1-64Q and MC3-64R, with one amino conversion of apo-mitrocomin into active photoprotein is a cause acid difference. of a low level of light emission. We tested the effect of Tyr deletion Among the 11 accidental substitutions per isolated mitrocomin on the apophotoprotein conversion into active mitrocomin. The re- cDNA genes (7390 bp in total) most of the random mutations (8 of 11, sults shown in Fig. 8 clearly demonstrate that the yield of an active ~72%) are transition mutations of A → Gtype,oneisG→ Atransition, truncated mitrocomin is ~2.5 times higher than that for the wild and two are transversions of A → T and T → A types from 12 possible type photoprotein. variants of single-nucleotide substitutions. Such mutation imbalance cannot arise as a result of DNA replication because mutations of A → G 4. Discussion type must appear on the opposite DNA chain as T → C mutations, i.e. the frequencies of transition mutations A → GandT→ Cshouldbe The eight different cDNA genes for Ca2+-regulated photoprotein equal if the mutation process is symmetric for both DNA strands. Thus, mitrocomin from a small tissue sample of the outer bell margin contain- the observed bias of mutation frequencies may be due to nucleotide ing photocytes of only one specimen of the jellyfish M. cellularia have misincorporations during asymmetric process of transcription, i.e. the been cloned. The highest heterogeneity was observed in the 3′-untrans- detected random mutations in mitrocomin cDNAs are the result of lated regions and was most likely a result of an imprecise cleavage after mRNA synthesis errors. Hence, three isolated mutant isoforms of polyadenylation signal, and/or an imprecise annealing of oligo(dT) mitrocomin MC3 are most likely the erroneous mRNA transcripts (Fig. primer (SMART 3′-NN-poly(T30)-5′) on any poly(A)-stretch in the 3′- 1) and, consequently, an inaccurate mRNA transcription might be one untranslated regions. All the isolated mitrocomin cDNAs fall into two of the possible sources of photoprotein isoform origin.

Table 3 Bioluminescence properties of mitrocomin and mitrocomin mutant with deletion of C-terminal tyrosine.

‡ Photoprotein Speciﬁc Bioluminescence, Fluorescence, kdecay Apparent dissociation constant (KD)of Apparent dissociation Speciﬁc −1 2+ 2+ bioluminescence λmax (nm) λmax (nm) (s ) apophotoprotein-coelenterazine complex constant (Kd) for Ca Ca -independent activity, (μM) (nM) luminescence (RLU/mg, ×1010) (RLU/mg, ×103)

MC2 118 ± 1.0 473 472 0.90 2.00 ± 0.01 117 ± 14 6.2 ± 0.04 MC2dY 184 ± 1.0 458 472 0.42 2.00 ± 0.08 172 ± 15 9.1 ± 0.01 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 295

Both newly isolated genomic isotypes MC1 and MC3 differ from the previously cloned isotype MI17 by 4 amino acid residues (Fig. S2 and S3). One position in amino acid sequences is variable for all of these isoforms – 64R/Q/K in isotypes MC1, MC3, and MI17, respectively. Note- worthy is that other hydromedusan photoproteins have Q or K in this position (Fig. 2), i.e. the residues which side chains contain amino group. Although the side chain of the residue in this position is found on the surface of photoprotein molecules, sufficiently conservative substitutions in this position may be due to the requirement of the photoprotein folding, for example. Beside mitrocomin, multiple photoprotein isotypes have been cloned for aequorin from A. victoria [12,13] and clytin from C. gregaria [16–18] as well as for light-sensitive photoprotein mnemiopsin from ctenophore Mnemiopsis leidyi [25–27]. Taking into account our findings we can reasonably suppose that some of these isotypes might be the result of transcriptional RNA mutations. Of note is that our efforts to clone isospecies of obelin from O. longissima [21,22] and berovin from ctenophore Beroe abyssicola failed [24]. The degree of sequence identities among the cloned photoprotein Fig. 7. Ca2+ concentration-effect curves for recombinant mitrocomin MC2 (squares) and its isotypes from the same jellyfish species vary approximately between mutant MC2dY with deletion of C-terminal Tyr (circles). Filled symbols, Ca-EGTA buffers; 0.5% (1 substitution) and 10%. For instance, aequorin isoform AEQ1 2+ open symbols, dilutions of CaCl2. L, light intensity at the particular Ca concentration, 2+ (M16103) shows 90.8% of sequence identity with A440 (L29571), clytin Lint, total light intensity at saturating Ca concentration. The measurements were performed at 20 °C. isotype CLI (CAA49754) reveals 91.5% of that with CL3 (GU721042), and mitrocomin MC3 displays 98% of the identity with MI17 (P39047). It should be pointed out that the amino acid sequence identity of The calculated mutation rate of ~1.5 × 10−3 misincorporations per photoproteins from the same genus almost matches the same range: base pair for the isolated mitrocomin cDNA genes is relatively high, 83.5–93.9% for aequorins, 72.7–93.9% for clytins, and 86% for obelins. but it well agrees with the one previously revealed for eukaryotic tran- The mitrocomin bioluminescence spectrum certainly differs from scription in vivo (~1.3 × 10−3/bp rate of substitutions) by Sanger se- those of other hydromedusan photoproteins. The recombinant obelins quencing of cDNA [63]. It should be noted that the transcriptional from O. geniculata and O. longissima have bioluminescence maxima at

fidelity in eukaryotic organisms is influenced by many factors [63,64] λmax = 485 and 495 nm, respectively, with a shoulder at 400 nm that and, consequently, the mutation rate can vary between representatives corresponds to the light emitted from the excited coelenteramide in of the same species. Hence, we cannot exclude that the determined its neutral state [1,9]. Although the bioluminescence maximum of clytin −3 error rate of ~1.5 × 10 misincorporations per base pair for the isolat- (λmax = 475 nm) [18] is very close to that of mitrocomin, mitrocomin ed mitrocomin cDNA genes can be characteristic of this specific speci- spectrum has no shoulder at 400 nm. The light emission spectrum of men having, for some reason, the reduced transcription fidelity. aequorin (λmax = 465 nm) [1,65] is shifted to shorter wavelengths for Thus, we believe that among the isolated isotypes of photoprotein almost 10 nm as compared to mitrocomin spectrum, however, it also mitrocomin only MC1 and MC3, differing in one amino acid (64R/Q) has no shoulder at 400 nm (Fig. 6A). are the products of two genome allelic genes. Most likely, other isolated Calcium binding to a photoprotein triggers the reaction to yield light isotypes MC2, MC4, and MC5 are the transcription mutants of MC3 emission, CO2, and the oxidation product (coelenteramide) tightly mitrocomin bearing additional substitutions Q109R, N40D/D135G, and bound to the apophotoprotein. Unlike the unreacted photoprotein, the E92G/Q176R, respectively (Fig. S2). This observation might exemplify Ca2+-discharged photoprotein efficiently fluoresces [1].Thefluores- the protein sequence diversity in organisms caused by transcription-as- cence spectra of Ca2+-discharged obelins from O. geniculata and O. sociated mutagenesis. longissima (λmax = 520 and 510 nm, respectively [23])andclytin (λmax =506nm[18]), for example, are shifted for about 25–30 nm to longer wavelengths as compared to their respective bioluminescence spectra. In contrast, the fluorescence spectra of Ca2+-discharged aequorin [1,65] and mitrocomin (Fig. 6A) are almost identical to their light emission spectra. Ca2+-regulated photoproteins are found in many different coelenterates, but the sequencing and structural information is presently available only for four different hydromedusan photoproteins: aequorin, obelin, clytin, and mitrocomin. These photoproteins show a high degree of identity in both amino acid sequences (Fig. 2) and spatial structures (Fig. 3). The cavity is formed by strictly conserved residues (Fig. 2). For instance, aequorin and mitrocomin differ from obelin and clytin in several positioins and it appears that one position particularly controls the bioluminescence colour [65]. In obelin and clytin, Phe is found respectively at sequence positions 88 and 91, whereas in aequorin and mitrocomin the corresponding positions are occupied by Tyr (Fig. 2). The role of the residue in this position in the photoprotein bioluminescence has been revealed by substitution of Phe to Tyr in obelin and Tyr to Phe in aequorin [65]. This substitution shifted obelin Fig. 8. Kinetics of conversion of MC2 (circles) and MC2dY (triangles) apo-mitrocomins bioluminescence to shorter wavelengths (λ = 453 nm), eliminating into active photoproteins monitored by bioluminescence. Experiments were carried out max in 5 mM EDTA, 10 mM DTT, 20 mM Tris-HCl pH 7.2 at 8 °C. Molar ratio for apoprotein/ the shoulder at 400 nm. In contrast, the same replacement in aequorin coelenterazine was 1:10. shifted its bioluminescence spectrum to λmax = 500 nm and led to the 296 L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 appearance of a shoulder at 400 nm, i.e. spectrum became resembling Additional Supporting Information may be found in the online ver- that of wild type obelin. It was suggested that the spectral differences sion of this article. Fig. S1 contains information about separation of the are due to an additional hydrogen bond in aequorin between the OH charged mitrocomin from apophotoprotein on Mono Q column; Fig. group of Tyr and the oxygen atom of the 6-(p-hydroxyphenyl) substit- S2 shows nucleotide alignment of eight full-size cDNA sequences of uent of coelenterazine [65]. Recently, spatial structures of two confor- mitrocomin genes isolated from one specimen of bioluminescent jelly- mation states (before and after bioluminescence reaction) of F88Y fish Mitrocoma cellularia (MC1, MC2, MC3, MC4, MC5, MC6, MC7, and obelin mutant have been determined [66]. A comparison of the hydro- MC8) as well as for the previously isolated MI17 cDNA gene; Fig. S3 rep- gen bond network formed by 2-hydroperoxycoelenterazine and resents a comparison of the five mitrocomin isotypes (MC1, MC2, MC3, coelenteramide with the key residues facing into the substrate-binding MC4, and MC5) isolated from cDNA library of the jellyfish Mitrocoma cavity of the wild type obelin and aequorin clearly shows that the main cellularia with the previously cloned MI17 isotype; Fig. S4 shows align- cause of different colors of light emission from hydromedusan ment of nucleotide sequences of four full-size cDNA genes encoding iso- photoproteins is a different arrangement of the hydrogen bond network forms of Ca2+-dependent coelenterazine-binding protein; Fig. S5 near an oxygen atom of 6-(p-hydroxyphenyl) group of coelenterazine represents a comparison of amino acid sequences of four isoforms of depending on the presence of either Phe or Tyr residue. coelenterazine-binding protein from R. muelleri. Supplementary data The hydrogen bond network formed by His24-Tyr90-Trp94 triad associated with this article can be found in the online version, at and oxygen atom of 6-(p-hydroxyphenyl) substituient in mitrocomin http://dx.doi.org. exactly corresponds to that of aequorin and F88Y obelin mutant (Fig. 4A). Thus, it clearly supports the conclusion resulting from spatial struc- Acknowledgments tures of two conformation states of F88Y obelin mutant, that such a type of hydrogen bond network arrangenment in this part of a substrate- We acknowledge the use of beamline 22-ID at the Advanced Photon binding cavity is responsible for the absence of a 400-nm shoulder in Source at Argonne National Laboratory (IL, USA). This work was sup- the photoprotein bioluminescence spectrum and also for the correspon- ported by the state budget allocated to the fundamental research at dence of fluorescence spectrum of Ca2+-discharged photoprotein to its the Russian Academy of Sciences (project No. 01201351504). This light emission spectrum. work was also supported by the CAS President's International Fellow- The mitrocomin amino acid sequence contains an additional Tyr res- ship Initiative (PIFI), National Natural Science Foundation of China idue at C-terminus as compared to other hydromedusan photoproteins (grant: 31330019), Ministry of Science and Technology of China (Fig. 2). In contrast to aequorin C-terminal Pro which deletion destroys (grant: 2014CB910400). bioluminescence function of a photoprotein [44,45] the removing of the C-terminal Tyr in mitrocomin even increases specific bioluminescence activity by 1.6 times and efficiency of conversion of apo-mitrocomin Appendix A. Supplementary data into active photoprotein. Why mitrocomin got the additional Tyr at C- terminus during evolution differing from other hydromedusan Supplementary data to this article can be found online at http://dx. photoproteins (Fig. 2) is a puzzle. However, we could formulate a cred- doi.org/10.1016/j.jphotobiol.2016.06.054. ible explanation based on mitrocomin nucleotide sequences. The Tyr residue is encoded by TAT or TAC codons whereas TAA, TGA, or TAG se- References quences code stop codons. If we assume that some ancestral gene for mitrocomin contained two successive stop codons as it is found in the [1] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientif- cDNA genes encoding Ca2+-dependent coelenterazine-binding protein ic, Singapore, 2006. [2] O. Shimomura, F.H. Johnson, Partial Purification and Properties of the Chaetopterus (CBP) from Renilla muelleri [67] (Fig. S4), for example, TAT codon for Luminescence System, in: F.H. Johnson, Y. Haneda (Eds.), Bioluminescence in Prog- Tyr may arise from stop codons TAA or TAG through a single-nucleotide ress, Princeton University Press, Princeton 1966, pp. 496–521. substitution either A → TorG→ T, respectively. Of note is that one of the [3] O. Shimomura, F.H. Johnson, Y. Saiga, Extraction, purification and properties of cloned CBP isoforms also has mutation in the first stop codon (Fig. S5) aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea,J. Cell. Comp. Physiol. 59 (1962) 223–239. leading to appearance of the additional amino acid residue (Fig. S5). [4] J.F. Head, S. Inouye, K. Teranishi, O. Shimomura, The crystal structure of the Since Tyr did not drastically influence bioluminescent function, most photoprotein aequorin at 2.3 Å resolution, Nature 405 (2000) 372–376. likely, this mutation survived during evolution. However, since the 3′- [5] Z.J. Liu, E.S. Vysotski, L. Deng, J. Lee, J.P. Rose, B.C. Wang, Atomic resolution structure of obelin: soaking with calcium enhances electron density of the second oxygen untranslated regions of hydromedusan genes are very variable Tyr res- atom substituted at the C2-position of coelenterazine, Biochem. Biophys. Res. idue may also appear as a result of random mutations. Commun. 311 (2003) 433–439. In summary, in the present study we have characterized several [6] M.S. Titushin, Y. Feng, G.A. Stepanyuk, Y. Li, S.V. Markova, S. Golz, B.C. Wang, J. Lee, J. 2+ Wang, E.S. Vysotski, Z.J. Liu, NMR-derived topology of a GFP-photoprotein energy isotypes of Ca -regulated photoprotein mitrocomin and described its transfer complex, J. Biol. Chem. 285 (2010) 40891–40900. crystal structure determined at 1.30 Å resolution. The analysis of cDNA [7] O. Shimomura, F.H. Johnson, Structure of the light-emitting moiety of aequorin, Bio- nucleotide sequences encoding mitrocomin isoforms allowed us to chemistry 11 (1972) 1602–1608. [8] M.J. Cormier, K. Hori, Y.D. Karkhanis, J.M. Anderson, J.E. Wampler, J.G. Morin, J.W. soundly suppose that two isoforms might be the products of two allelic Hastings, Evidence for similar biochemical requirements for bioluminescence genes whereas other isotypes appear as a result of transcriptional muta- among the coelenterate, J. Cell. Physiol. 81 (1973) 291–297. tions and it might be the cause of diversity of other hydromedusan [9] E.S. Vysotski, J. Lee, Ca2+-regulated photoproteins: structural insight into the bioluminescence mechanism, Acc. Chem. Res. 37 (2004) 405–415. photoproteins as well. We have also demonstrated that various [10] J.G. Morin, Coelenterate Bioluminescence, in: L. Muscatine, H.M. Lenhoff (Eds.), Coe- mitrocomin isoforms have different specific bioluminescence activities, lenterate Biology: Reviews and New Perspectives, Academic Press, New York 1974, but their spectral properties are the same. In addition, using site-direct- pp. 397–438. ed mutagenesis, we have shown that C-terminal Tyr is not crucially im- [11] D. Prasher, R.O. McCann, M.J. Cormier, Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein, Biochem. Biophys. Res. portant for bioluminescence function because its deletion even Commun. 126 (1985) 1259–1268. increases the specific bioluminescence activity of mitrocomin and the [12] S. Inouye, M. Noguchi, Y. Sakaki, Y. Takagi, T. Miyata, S. Iwanaga, T. Miyata, F.I. Tsuji, fi Cloning and sequence analysis of cDNA for the luminescent protein aequorin, Proc. ef ciency of conversion of apo-mitrocomin into an active photoprotein. – fi Natl. Acad. Sci. U. S. A. 82 (1985) 3154 3158. Based on our ndings and the fact that genes in a population are gener- [13] D.C. Prasher, R.O. McCann, M. Longiaru, M.J. Cormier, Sequence comparisons of com- ally present as different isoforms, we can also assume that there are plementary DNAs encoding aequorin isotypes, Biochemistry 26 (1987) 1326–1332. even more isoforms of mitrocomin, and of other photoproteins as [14] N.G. Gurskaya, A.F. Fradkov, N.I. Pounkova, D.B. Staroverov, M.E. Bulina, Y.G. Yanushevich, Y.A. Labas, S. Lukyanov, K.A. Lukyanov, A colourless green fluorescent well, which can be cloned in future and which might differ in biolumi- protein homologue from the nonfluorescent hydromedusa Aequorea coerulescens nescence properties. and its fluorescent mutants, Biochem. J. 373 (2003) 403–408. L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 297

[15] N.S. Xia, W.X. Luo, J. Zhang, X.Y. Xie, H.J. Yang, S.W. Li, M. Chen, M.H. Ng, Biolumines- [41] V.P. Koldenkova, T. Nagai, Genetically encoded Ca2+ indicators: properties and eval- cence of Aequorea macrodactyla, a common jellyfish species in the East China Sea, uation, Biochim. Biophys. Acta 1833 (2013) 1787–1797. Mar. Biotechnol. 4 (2002) 155–162. [42] F.I. Tsuji, Y. Ohmiya, T.F. Fagan, H. Toh, S. Inouye, Molecular evolution of the Ca2+- [16] S. Inouye, F.I. Tsuji, Cloning and sequence analysis of cDNA for the Ca2+-activated binding photoproteins of the hydrozoa, Photochem. Photobiol. 62 (1995) 657–661. photoprotein, clytin, FEBS Lett. 315 (1993) 343–346. [43] E.S. Vysotski, S.V. Markova, L.A. Frank, Calcium-regulated photoproteins of marine [17] S. Inouye, Cloning, expression, purification and characterization of an isotype of coelenterates, Mol. Biol. 40 (2006) 355–367. clytin, a calcium-binding photoprotein from the luminous hydromedusa Clytia [44] M. Nomura, S. Inouye, Y. Ohmiya, F.I. Tsuji, A C-terminal proline is required for bio- gregarium, J. Biochem. 143 (2008) 711–717. luminescence of the Ca2+-binding photoprotein, aequorin, FEBS Lett. 295 (1991) [18] S.V. Markova, L.P. Burakova, L.A. Frank, S. Golz, K.A. Korostileva, E.S. Vysotski, Green- 63–66. fluorescent protein from the bioluminescent jellyfish Clytia gregaria:cDNAcloning, [45] N.J. Watkins, A.K. Campbell, Requirement of the C-terminal proline residue for sta- expression, and characterization of novel recombinant protein, Photochem. bility of the Ca2+-activated photoprotein aequorin, Biochem. J. 293 (1993) 181–185. Photobiol. Sci. 9 (2010) 757–765. [46] S.K. Deo, J.C. Lewis, S. Daunert, C-terminal and N-terminal fusions of aequorin with [19] C. Fourrage, K. Swann, J.R. Gonzalez Garcia, A.K. Campbell, E. Houliston, An endoge- small peptides in immunoassay development, Bioconjug. Chem. 12 (2001) nous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs 378–384. shows co-targeting to mitochondria and efficient bioluminescence energy transfer, [47] E.V. Eremeeva, L.A. Frank, S.V. Markova, E.S. Vysotski, Ca2+-regulated photoprotein Open Biol. 4 (2014) 130206. obelin as N-terminal partner in the fusion proteins, J. Sib. Fed. Univ., Biol. 4 (2010) [20] T.F. Fagan, Y. Ohmiya, J.R. Blinks, S. Inouye, F.I. Tsuji, Cloning, expression and se- 372–383. quence analysis of cDNA for the Ca2+-binding photoprotein, mitrocomin, FEBS [48] S.V. Markova, E.S. Vysotski, Coelenterazine-dependent luciferases, Biochemistry Lett. 333 (1993) 301–305. (Mosc) 80 (2015) 714–732. [21] B.A. Illarionov, S.V. Markova, V.S. Bondar, E.S. Vysotski, J.I. Gitelson, Cloning and ex- [49] B.A. Illarionov, L.A. Frank, V.A. Illarionova, V.S. Bondar, E.S. Vysotski, J.R. Blinks, Re- pression of cDNA for the Ca2+-activated photoprotein obelin from the hydroid combinant obelin: cloning and expression of cDNA, purification and characteriza- polyp Obelia longissima, Dokl. Akad. Nauk 326 (1992) 911–913. tion as a calcium indicator, Methods Enzymol. 227 (2000) 223–249. [22] B.A. Illarionov, V.S. Bondar, V.A. Illarionova, E.S. Vysotski, Sequence of the cDNA [50] E.S. Vysotski, Z.J. Liu, J. Rose, B.C. Wang, J. Lee, Preparation and X-ray crystallographic encoding the Ca2+-activated photoprotein obelin from the hydroid polyp Obelia analysis of recombinant obelin crystals diffracting to beyond 1.1 Å, Acta Crystallogr. longissima, Gene 153 (1995) 273–274. D Biol. Crystallogr. 57 (2001) 1919–1921. [23] S.V. Markova, E.S. Vysotski, J.R. Blinks, L.P. Burakova, B.C. Wang, J. Lee, Obelin from [51] E.V. Eremeeva, S.V. Markova, W.J. van Berkel, E.S. Vysotski, Role of key residues of the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and com- obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct, J. parison of some properties with those of other Ca2+-regulated photoproteins, Bio- Photochem. Photobiol. B 127 (2013) 133–139. chemistry 41 (2002) 2227–2236. [52] N.P. Malikova, L.P. Burakova, S.V. Markova, E.S. Vysotski, Characterization of [24] S.V. Markova, L.P. Burakova, S. Golz, N.P. Malikova, L.A. Frank, E.S. Vysotski, The hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+ light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe concentration, Anal. Bioanal. Chem. 406 (2014) 5715–5726. abyssicola:anoveltypeofCa2+-regulated photoprotein, FEBS J. 279 (2012) [53] N.P. Malikova, A.J. Borgdorff, E.S. Vysotski, Semisynthetic photoprotein reporters for 856–870. tracking fast Ca2+ transients, Photochem. Photobiol. Sci. 14 (2015) 2213–2224. [25] M.R. Aghamaali, V. Jafarian, R. Sariri, M. Molakarimi, B. Rasti, M. Taghdir, R.H. Sajedi, [54] M. Klabusay, J.R. Blinks, Some commonly overlooked properties of calcium buffer S. Hosseinkhani, Cloning, sequencing, expression and structural investigation of systems: a simple method for detecting and correcting stoichiometric imbalance mnemiopsin from Mnemiopsis leidyi: an attempt toward understanding Ca2+-regu- in CaEGTA stock solutions, Cell Calcium 20 (1996) 227–234. lated photoproteins, Protein J. 30 (2011) 566–574. [55] D.G. Allen, J.R. Blinks, F.G. Prendergast, Aequorin luminescence: relation of light [26] V. Jafarian, R. Sariri, S. Hosseinkhani, M.R. Aghamaali, R.H. Sajedi, M. Taghdir, S. emission to calcium concentration – a calcium-independent component, Science Hassannia, A unique EF-hand motif in mnemiopsis photoprotein from Mnemiopsis 195 (1977) 996–998. leidyi: implication for its low calcium sensitivity, Biochem. Biophys. Res. Commun. [56] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation 413 (2011) 164–170. mode, Methods Enzymol. 276 (1997) 307–326. [27] C.E. Schnitzler, K. Pang, M.L. Powers, A.M. Reitzel, J.F. Ryan, D. Simmons, T. Tada, M. [57] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Park, J. Gupta, S.Y. Brooks, R.W. Blakesley, S. Yokoyama, S.H. Haddock, M.Q. Phaser crystallographic software, J. Appl. Cryst. 40 (2007) 658–674. Martindale, A.D. Baxevanis, Genomic organization, evolution, and expression of [58] P.D. Adams, P.V. Afonine, G. Bunkóczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.- photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, photocytes, BMC Biol. 10 (2012) 107. R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, P.H. Zwart, PHENIX: a [28] M.L. Powers, A.G. McDermott, N.C. Shaner, S.H. Haddock, Expression and character- comprehensive python-based system for macromolecular structure solution, Acta ization of the calcium-activated photoprotein from the ctenophore Bathocyroe Crystallogr. D Biol. Crystallogr. 66 (2010) 213–221. fosteri: insights into light-sensitive photoproteins, Biochem. Biophys. Res. Commun. [59] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta 431 (2013) 360–366. Crystallogr. D Biol. Crystallogr. 60 (2004) 2126–2132. [29] O. Shimomura, F.H. Johnson, Regeneration of the photoprotein aequorin, Nature 256 [60] Z.J. Liu, E.S. Vysotski, C.J. Chen, J.P. Rose, J. Lee, B.C. Wang, Structure of the Ca2+-reg- (1975) 236–238. ulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur [30] E.V. Eremeeva, S.V. Markova, A.H. Westphal, A.J. Visser, W.J. van Berkel, E.S. Vysotski, substructure, Protein Sci. 11 (2000) 2085–2093. The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching [61] O. Shimomura, Isolation and properties of various molecular forms of aequorin, to characterize coelenterazine binding, FEBS Lett. 583 (2009) 1939–1944. Biochem. J. 234 (1986) 271–277. [31] E.V. Eremeeva, P.V. Natashin, L. Song, Y. Zhou, W.J. van Berkel, Z.J. Liu, E.S. Vysotski, [62] Y.S. Vysotskii, V.S. Bondar’, I.I. Gitelzon, Isolation and properties of different molecu- Oxygen activation of apo-obelin-coelenterazine complex, Chembiochem 14 (2013) lar forms of calcium-activated photoprotein obelin, Doklady Akad. Nauk SSSR 321 739–745. (1991) 214–217. [32] E.B. Ridgway, C.C. Ashley, Calcium transients in single muscle fibers, Biochem. [63] N.K. Nesser, D.O. Peterson, D.K. Hawley, RNA polymerase II subunit Rpb9 is impor- Biophys. Res. Commun. 29 (1967) 229–234. tant for transcriptional fidelity in vivo, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) [33] J.R. Blinks, W.G. Wier, P. Hess, F.G. Prendergast, Measurement of Ca2+ concentra- 3268–3273. tions in living cells, Prog. Biophys. Mol. Biol. 40 (1982) 1–114. [64] H. Koyama, T. Ueda, T. Ito, K. Sekimizu, Novel RNA polymerase II mutation sup- [34] R.Y. Tsien, Intracellular measurements of ion activities, Annu. Rev. Biophys. Bioeng. presses transcriptional fidelity and oxidative stress sensitivity in rpb9Delta yeast, 12 (1983) 91–116. Genes Cells 15 (2010) 151–159. [35] J.D. Bruton, A.J. Cheng, H. Westerblad, Methods to detect Ca2+ in living cells, Adv. [65] G.A. Stepanyuk, S. Golz, S.V. Markova, L.A. Frank, J. Lee, E.S. Vysotski, Interchange of Exp. Med. Biol. 740 (2012) 27–43. aequorin and obelin bioluminescence color is determined by substitution of one ac- [36] M. Whitaker, Genetically encoded probes for measurement of intracellular calcium, tive site residue of each photoprotein, FEBS Lett. 579 (2005) 1008–1014. Methods Cell Biol. 99 (2010) 153–182. [66] P.V. Natashin, S.V. Markova, J. Lee, E.S. Vysotski, Z.J. Liu, Crystal structures of the F88Y [37] D. Ottolini, T. Calì, M. Brini, Measurements of Ca2+ concentration with recombinant obelin mutant before and after bioluminescence provide molecular insight into targeted luminescent probes, Methods Mol. Biol. 937 (2013) 273–291. spectral tuning among hydromedusan photoproteins, FEBS J. 281 (2014) [38] M. Bonora, C. Giorgi, A. Bononi, S. Marchi, S. Patergnani, A. Rimessi, R. Rizzuto, P. 1432–1445. Pinton, Subcellular calcium measurements in mammalian cells using jellyfish [67] M.S. Titushin, S.V. Markova, L.A. Frank, N.P. Malikova, G.A. Stepanyuk, J. Lee, E.S. photoprotein aequorin-based probes, Nat. Protoc. 8 (2013) 2105–2118. Vysotski, Coelenterazine-binding protein of Renilla muelleri:cDNAcloning,overex- [39] V. Baubet, H. LeMouellic, A.K. Campbell, E. Lucas-Meunier, P. Fossier, P. Brulet, Chi- pression, and characterization as a substrate of luciferase, Photochem. Photobiol. meric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the Sci. 7 (2008) 189–196. single-cell level, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7260–7265. [40] A. Bakayan, B. Domingo, A. Miyawaki, J. Llopis, Imaging Ca2+ activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant, Pflugers Arch. 467 (2015) 2031–2042.