Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins Pavel V. Natashin1,2,3, Svetlana V. Markova2,3, John Lee4, Eugene S. Vysotski2,3 and Zhi-Jie Liu1,5 1 2 3 4 5

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia Laboratory of Bioluminescence Biotechnology, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Russia Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA iHuman Institute, ShanghaiTech University, Shanghai, China

Keywords aequorin; bioluminescence; Ca2+-regulated photoprotein; coelenterazine, obelin Correspondence Z.-J. Liu, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Fax: +86 10 64888426 Tel: +86 10 64888426 E-mail: [email protected] E. S. Vysotski, Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Akademgorodok 50, Building 50, Krasnoyarsk 660036, Russia Fax: +7 391 243 3400 Tel: +7 391 249 4430 E-mail: [email protected] (Received 16 October 2013, revised 15 December 2013, accepted 4 January 2014) doi:10.1111/febs.12715

Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2-hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462–495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008–1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca2+ discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

Introduction Bioluminescence is a widely distributed phenomenon among marine dwellers [1,2], many of which generate light by oxidation of coelenterazine, an imidazopyrazinone derivative [3,4]. The chemical mechanism of light emission appears to be common for various organisms that utilize coelenterazine, but often differs in the detailed biochemical process, probably as necessitated by the behavioral function of bioluminescence [5]. 1432

Ca2+-regulated photoproteins constitute a unique class of protein biochemistry. They are responsible for the light emission of a variety of marine coelenterates [3]. The best known and studied of these photoproteins are aequorin, first isolated from the jellyfish Aequorea victoria [6], and obelin, from the hydroid Obelia longissima [7]. Ca2+-regulated photoproteins are ‘pre-charged’ bioluminescent proteins that are FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

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triggered to emit light by binding Ca2+ or certain other inorganic ions [8]. The reaction does not require the presence of molecular oxygen or any other cofactor; the photoprotein and the triggering ion are the only components required for light emission. As the energy emitted as light is derived from the ‘charged’ photoprotein, that molecule may react only once, i.e. it does not ‘turn over’ as an enzyme does. In this respect, as well as with regard to the lack of a requirement for molecular oxygen or any other cofactor, the reaction is strikingly different from that of classical bioluminescent systems in which an enzyme (luciferase) catalyzes oxidation of a smaller organic substrate molecule (luciferin) resulting in creation of an excited state and the emission of light. This feature prompted Shimomura and Johnson to coin the term ‘photoprotein’ to describe proteins that contain a reactive organic molecule in their bioluminescent reaction system [9]. Although they described another kind of photoprotein, the great majority of photoproteins presently known are stimulated to luminescence by calcium ions, and the term ‘Ca2+-activated photoproteins’ was applied to them by Hastings and Morin [10]. Later, the term ‘Ca2+-regulated photoproteins’ was suggested for this group, first because these proteins are the members of the family of Ca2+-regulated proteins, and second because calcium regulates the bioluminescence function of these proteins but is not essential for it [11]. Ca2+free photoproteins emit a very low level of light named ‘Ca2+-independent bioluminescence’, but the light intensity increases one million-fold or even more after addition of calcium [12]. All Ca2+-regulated photoproteins consist of a single polypeptide chain with a molecular mass of ~ 22 kDa, to which the oxygen-activated substrate, 2-hydroperoxycoelenterazine, is tightly but non-covalently bound. The bioluminescence reaction involves oxidative breakdown of the substrate to the S1 excited state of the product, coelenteramide [13,14]. The bioluminescence has a broad spectral distribution with a maximum that depends on the photoprotein type [15]. Although Ca2+-regulated photoproteins have apparently been detected in many (> 25) coelenterates [3], cloning and sequence analysis have been achieved only for five hydromedusan photoproteins, i.e. aequorin from A. victoria [16–18], clytin (also known as phialidin) from Clytia gregaria [19–21], mitrocomin (also known as halistaurin) from Mitrocoma cellularia [22], and obelins from O. longissima [23,24] and O. geniculata [15], and for light-sensitive photoproteins from the ctenophores Beroe abyssicola [25], Mnemiopsis leidyi [26,27] and Bathocyroe fosteri [28]. Although hydromedusan and ctenophore photoproteins are functionally FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

Structures of F88Y obelin mutant in two states

identical in many respects, contain three calcium-binding sites characteristic of EF-hand Ca2+-binding proteins, and possess the same spatial architecture [29], the degree of identity of their amino acid sequences is only ~ 29% [25,28]. Apophotoproteins can be converted into active photoproteins by incubating them with coelenterazine under calcium-free conditions in the presence of O2 and reducing agents [30]. Ca2+-regulated photoproteins draw a strong interest due to their wide analytical potential. The main use of photoproteins derives from their ability to emit light upon Ca2+ binding, and they have been used to detect calcium ions in biological systems [31]. The cloning of cDNAs encoding apophotoproteins has opened new avenues for utilizing photoproteins, by expressing the recombinant apophotoprotein intracellularly, then adding coelenterazine externally, which diffuses into the cell and forms the active photoprotein. In effect, such cells have a ‘built-in’ calcium indicator. This approach is highly effective and is widely applied [32–34]. Over the past decade, the crystal structures of the hydromedusan photoproteins aequorin, obelin and clytin, as well as obelin ligand-dependent conformation states and obelin mutant with altered spectral properties, have been determined [35–43]. These findings and mutagenesis studies of the function of residues that form the substrate-binding cavity [44–48] have allowed the proposal of a proton-relay mechanism for triggering of a bioluminescence reaction by Ca2+ and for formation of different product excited states, as well as the function of some residues in these processes [49,50]. All photoproteins have the same globular structure formed by the N- and C-terminal domains, each comprising two helix-turn-helix motifs known as EF-hands that are characteristic of the superfamily of EF-hand Ca2+-binding proteins [51]. The substratebinding cavities are highly hydrophobic, and are formed by practically the same residues in each photoprotein (Fig. 1). In addition, the hydrophilic side chains of two His and two Tyr residues are also directed into the cavity. All these residues are present within hydrogen bond distances of the atoms of 2-hydroperoxycoelenterazine, and significantly influence the bioluminescence function of photoproteins. For example, the side chains of the His175-Tyr190-Trp179 triad in obelin (Fig. 1) and the same residues in other photoproteins are in close proximity with the peroxy and carbonyl groups of 2-hydroperoxycoelenterazine, implying formation of strong hydrogen bonds between them. It was suggested that this triad participates in stabilization of the 2-hydroperoxycoelenterazine molecule within internal photoprotein cavity [31,33], and that this His residue may be the key residue for trig1433

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Structures of F88Y obelin mutant in two states

Fig. 1. Sequence alignment of obelin from Obelia longissima [24], clytin from Clytia gregaria [21], aequorin from Aequorea victoria [17] and mitrocomin from Mitrocoma cellularia [22]. The mutation in obelin is shown in blue. Strictly conserved residues of the substrate-binding pocket are shown in red. Variable residues found in the substratebinding pocket are shown in green. The loops involved in Ca2+ binding are highlighted in yellow. The helices are indicated by capital letters A–H based on the WT obelin structure (PDB code 1QV0).

gering bioluminescence by Ca2+ [41,50] and formation of an active photoprotein complex from apophotoprotein, coelenterazine and O2 [52,53]. Although the substrate-binding cavities of hydromedusan photoproteins are formed by strictly conserved residues (Fig. 1), their bioluminescence spectra are different. The recombinant obelins from O. geniculata and O. longissima have bioluminescence maxima (kmax) at 485 and 495 nm, respectively, with a shoulder at 400 nm [15] that corresponds to light emitted from the excited coelenteramide in its neutral state [8]. The light emission spectra of aequorin (kmax = 462–469 nm [8,47]) and mitrocomin (kmax = 470 nm [22]) are shifted to shorter wavelengths compared to obelins and have no shoulder at 400 nm. Although the bioluminescence maximum of clytin (kmax = 475 nm [21]) is very close to that of mitrocomin, the clytin bioluminescence spectrum has a shoulder at 400 nm like obelins. Aequorin and mitrocomin differ from obelin and clytin at four and three positions, respectively (Fig. 1), and it appears, that one position in particular controls the bioluminescence color [47]. In obelin and clytin, Phe is found at positions 88 and 91, respectively, whereas, in aequorin and mitrocomin, the corresponding positions are occupied by Tyr (Fig. 1). The effect of the residue in this position on the photoprotein bioluminescence has been revealed by substitution of Phe to Tyr in obelin and Tyr to Phe in aequorin [47]. Substitution shifted the obelin bioluminescence to a shorter wavelength (kmax = 453 nm) and eliminated the shoulder at 400 nm, thus making bioluminescence spectrum of obelin similar to that of wild-type (WT) aequorin (kmax = 469 nm). In contrast, the substitution in aequorin shifted its bioluminescence spectrum to kmax = 500 nm, and initiated the appearance of a shoulder at 400 nm, i.e. produced the emission spectrum resembling that of WT obelin. It was suggested 1434

that the spectral differences are due to an additional hydrogen bond in aequorin between the hydroxyl group of Tyr and the oxygen atom of the 6-(p-hydroxyphenyl) group of coelenterazine [47,50]. In the present study, we report the crystal structures of the F88Y obelin mutant before and after the biolu resolutions, minescence reaction at 2.09 and 1.50 A respectively. In addition to mutagenesis studies, these spatial structures provide a rational molecular basis to explain the spectral differences between hydromedusan photoproteins.

Results Overall structure The spatial structures of F88Y obelin and its Ca2+discharged conformation state retain the same overall scaffold that is characteristic of different ligand-dependent conformation states of Ca2+-regulated photoproteins [35–37,39–41], and both structures closely resemble those of the corresponding conformation states of WT obelin [37,41] (Fig. 2A,B). Crystal structures of the active F88Y obelin mutant, i.e. bound to 2-hydroperoxycoelenterazine, and in its Ca2+-discharged form, i.e. bound to the bioluminescence reaction product coelenteramide and calcium ions, contain one and two molecules per asymmetric unit, respectively. The structures of WT obelin and its F88Y mutant show almost no difference except for the orientations of some surface side chains. The RMSD of the mainand side-chain atoms for these proteins is only 0.36  respectively (Table 1). However, the difand 1.06 A, ference between Ca2+-discharged F88Y and WT obelins is much greater (the RMSD of main-/side-chain  but almost all large differences atoms is 1.71/2.65 A), FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

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Structures of F88Y obelin mutant in two states

A

Fig. 2. (A,B) Overall spatial structures of F88Y obelin (A) and its Ca2+-discharged conformation state (B). The crystal structure of Ca2+-discharged F88Y obelin contains two molecules per asymmetric unit. (C) Stereoview of superimposition of F88Y obelin (yellow), Ca2+-discharged F88Y obelin (blue), WT obelin (red), Ca2+discharged WT obelin (green), and WT aequorin (cyan). The 2hydroperoxycoelenterazine and coelenteramide molecules are indicated by the stick models in the center of the protein; the calcium ions are indicated by balls. The 2-hydroperoxycoelenterazine, coelenteramide, and calcium ions are colored according to the structure color.

B

C

Table 1. Comparison of conformation states of F88Y obelin.

Structural parts of photoprotein Overallc N-terminal domaind C-terminal domain EF-hand motif Ie EF-hand motif II EF-hand motif III EF-hand motif IV

F88Y obelin versus Ca2+-discharged F88Y obelinb

F88Y obelin versus WT obelin

Ca2+-discharged F88Y obelin versus Ca2+discharged WT obelin

F88Y obelin versus WT aequorin

0.36/1.06 0.31/1.15 0.34/0.90 0.24/1.02 0.25/0.99 0.39/1.16 0.25/0.82

1.71/2.65 0.50/1.54 2.37/3.53 0.35/1.35 0.20/1.19 2.17/3.66 0.84/2.03

0.78 0.82 0.62 1.05 0.45 0.72 0.39

RMSD values ( A)a 2.24/3.11 1.23/2.12 2.82/3.87 1.42/2.27 0.49/1.56 2.07/3.14 2.05/3.68

a

RMSD values were calculated using the program Superpose Molecules (CCP4) [67]. Values are for main-/side-chain atoms for obelins and main-chain atoms for aequorin. b The comparison was performed for molecule A of Ca2+-discharged F88Y obelin. c Residues 9–195 for F88Y and WT obelins; residues and 9–191 for Ca2+-discharged F88Y and WT obelins; residues 3–189 for aequorin. d N- and C-terminal domains are defined as residues 9–106 and 107–195, respectively, for WT and F88Y obelins, residues 9–106 and 107– 195, respectively, for Ca2+-discharged WT and F88Y obelins, and residues 3–100 and 101–189, respectively, for aequorin. e EF-hand motifs I, II, III and IV are defined as residues 17–54, 58–104, 110–141 and 149–180, respectively, for obelins, and residues 11–48, 52–98, 95–135 and 143–174, respectively, for aequorin.

are found in EF-hand motif III of the C-terminal domain. The RMSD of the main-/side-chain atoms of F88Y obelin versus its Ca2+-discharged conformation  Although these values exceed state is 2.24/3.11 A. those for the corresponding states of WT obelin [41], the conformation of F88Y obelin undergoes changes FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

in response to Ca2+ binding that are very similar to those in WT obelin. The deviations in the C-terminal domain and EF-hand motif IV are greater than in the N-terminal domain and other EF-hand motifs, and EF-hand motif II changes insignificantly in both WT and mutant obelins [41]. It should be noted that the 1435

Structures of F88Y obelin mutant in two states

RMSD value of the main chain atoms of F88Y obelin  This value is very close versus WT aequorin is 0.78 A.  to that for WT obelin versus WT aequorin (0.80 A). The structures of all calcium-binding loops and any changes occurring therein after the reaction in F88Y obelin are essentially the same as in WT obelin [41], and therefore are not shown. In Ca2+-discharged F88Y obelin, calcium ions are found in Ca2+-binding loops I, III and IV (Fig. 2B). In all Ca2+-binding loops, the calcium ion is coordinated in a pentagonal  separation to oxygen bi-pyramidal array with ~ 2.4 A 2+ atoms. Similar to Ca -discharged obelin [41], six oxygen ligands derive from the side chains of Asp and Glu, Asn or Ser, the carbonyl groups of the peptide backbone, and the seventh oxygen ligand comes from water molecule. These spatial structure parameters (Table 1) show that substitution of Phe to Tyr does not significantly affect the overall structure and structural rearrangements in the photoprotein molecule in response to Ca2+ binding.

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Hydrogen bond network in the substrate-binding cavity of F88Y obelin before and after bioluminescence Figure 3 shows the hydrogen bond network formed by the 2-hydroperoxycoelenterazine atoms and some key surrounding residues in F88Y obelin. His22 and Tyr88 are hydrogen-bonded with the oxygen of the 6-(p-hydroxyphenyl) group of coelenterazine, and the Ne of Trp92 forms a hydrogen bond with the hydroxyl group of Tyr88. The hydroxyl group of Tyr190 has a strong hydrogen bond to the hydroperoxide group, His175 forms hydrogen bonds with Tyr190, the Ne of Trp179 has a possible weak hydrogen bond to the C3 carbonyl oxygen, and the oxygen of Tyr138 hydroxyl group forms a hydrogen bond with N1 of 2-hydroperoxycoelenterazine. There are also two water molecules (W1 and W2) that are stabilized by hydrogen bonds with the surrounding residues and the OH of the 2-(p-hydroxybenzyl) substituent of coelenterazine. A

A

B

Fig. 3. Two-dimensional drawing of the hydrogen bond network (A), and stereoview of the 2-hydroperoxycoelenterazine molecule with the key residues facing into the substrate-binding cavity (B), for F88Y obelin. Hydrogen bonds are indicated by dashed lines (A) and dots (B). Distances are given in  A. Water molecules are indicated by cyan-colored balls.

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Structures of F88Y obelin mutant in two states

third water molecule (W3) is found close to the surface of the photoprotein molecule and is stabilized by hydrogen bonds with His64, Ser47 and Thr61. Thus, there is a noticeable difference between the hydrogen bond network of F88Y obelin (Fig. 3) and WT obelin (Fig. 4); in WT obelin, the oxygen of the 6-(p-hydroxyphenyl) group of coelenterazine is hydrogen-bonded only with His22. Although the Ne of Trp92 resides  (Fig. 4), the PyMOL proclose to this oxygen (3.26 A) gram does not reveal a hydrogen bond, perhaps due to the constrains for angles between atoms at the formation of hydrogen bonds. In addition, while in F88Y obelin, the Ne of Trp114 has a possible weak hydrogen bond to W2 (Fig. 3A), this hydrogen bond is not found at all in WT obelin (Fig. 4). Figure 5 shows the hydrogen bond network in the substrate-binding cavity of aequorin. The hydrogen bond pattern in aequorin is almost identical to that of F88Y obelin. There is one apparent difference: in

F88Y obelin, Trp179 forms a hydrogen bond with the 2-hydroperoxycoelenterazine carbonyl oxygen, whereas in aequorin, the corresponding hydrogen bond from Trp173 is absent. It is noteworthy that the aequorin hydrogen bond network formed by the His16-Tyr82Trp86 triad is identical to that of F88Y obelin (His22Tyr88-Trp92 triad), and shows identical differences from WT obelin to those observed between F88Y and WT obelins. The hydrogen bond network formed by coelenteramide in Ca2+-discharged F88Y obelin is shown in Fig. 6. After the bioluminescence reaction, His22 and Tyr88 are found in the same positions and retain hydrogen bonds with the oxygen atom of the 5-(p-hydroxyphenyl) group of coelenteramide, and Trp92 is still hydrogen-bonded with Tyr88, i.e. the hydrogen bond pattern is identical to that before bioluminescence. It should be pointed out that, in Ca2+-discharged WT obelin, a new but weak hydrogen bond between Trp92

A

B

Fig. 4. Two-dimensional drawing of the hydrogen bond network (A), and stereoview of 2-hydroperoxycoelenterazine molecule with the key residues facing into the substrate-binding cavity (B), for WT obelin (PDB code 1QV0). Hydrogen bonds are indicated by dashed lines (A) and dots (B). Distances are given in  A. Water molecules are indicated by cyan-colored balls.

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A

B

Fig. 5. Two-dimensional drawing of the hydrogen bond network (A), and stereoview of 2-hydroperoxycoelenterazine molecule with the key residues facing into the substrate-binding cavity (B), for aequorin (PDB code 1EJ3). Hydrogen bonds are indicated by dashed lines (A) and dots (B). Distances are given in  A. Water molecules are indicated by cyancolored balls.

and the oxygen of the 5-(p-hydroxyphenyl) group of coelenteramide is detected (Fig. 7). The Ne of Trp179 that previously hydrogen-bonded with the C3 carbonyl oxygen (Fig. 3) forms a new hydrogen bond with Tyr190 after the reaction. Tyr190, which previously stabilized the 2-hydroperoxy group of coelenterazine by a hydrogen bond and was also hydrogen-bonded to His175, misses these hydrogen bonds after the reaction but forms a new bond with the carbonyl oxygen of coelenteramide. His175 also forms a new hydrogen bond with water molecule W1, which is apparently repositioned as a result of the changed position of the 2-(p-hydroxybenzyl) group of coelenterazine. W1 only retains hydrogen bonds with the carbonyl oxygen of Ile111 and the oxygen of the 2-(p-hydroxybenzyl) substituent, and 1438

forms a new bond with His175. Thus, the changes occurring in this part of the substrate-binding cavity in response to Ca2+ binding and following decarboxylation of coelenterazine are identical to those taking place after Ca2+ discharge of WT obelin (Fig. 7). The oxygen of Tyr138 originally hydrogen bonded with N1 of 2-hydroperoxycoelenterazine (Fig. 3) is replaced by water molecule W2 (Fig. 6), which apparently connected Tyr138 with His64 before the reaction. His64 is also slightly shifted toward coelenteramide, retaining its hydrogen bond with W2. In addition, W2, while preserving a hydrogen bond with Trp114, also forms a new hydrogen bond with the carbonyl oxygen of Ala46 that appears in the cavity after the reaction. As His64 is shifted, water molecule W3 retains a hydrogen bond FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

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Structures of F88Y obelin mutant in two states

A

B

Fig. 6. Two-dimensional drawing of the hydrogen bond network (A), and stereoview of the coelenteramide molecule with the key residues facing into the substrate-binding cavity (B), for Ca2+discharged F88Y obelin. Hydrogen bonds are indicated by dashed lines (A) and dots (B). Distances are given in  A. Water molecules are indicated by cyan-colored balls.

with Ser47 and forms new ones with the oxygen atoms of Asp48 and Gln65 (Fig. 6). Although the changes in the hydrogen bond network in this region look very similar in Ca2+-discharged F88Y and WT obelins, there is a critical difference. While the Tyr138 in Ca2+-discharged WT obelin moves to the surface of the photoprotein molecule [41], this residue remains within the cavity in Ca2+-discharged F88Y obelin, forming a hydrogen bond with water molecule W4, which is also situated within the protein globule but at a distance  from coelenteramide atoms that slightly exceeds 4 A (Fig. 6). Additionally, W4 forms two hydrogen bonds with Met171 and Gln174.

Discussion Determination of the spatial structures of various hydromedusan Ca2+-regulated photoproteins [35–37] and their different conformation states [39–41], as well as the obelin mutant with altered spectral properties FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

[42,43], and comprehensive mutagenesis studies of the function of residues forming the substrate-binding cavity [44–48], have allowed proposal of a proton-relay mechanism for triggering of a bioluminescence reaction by Ca2+ and for formation of different product excited states [49,50]. According to the suggested mechanism, the neutral coelenteramide is the primary excited product in photoprotein bioluminescence, and coelenteramide excited state that emits light at longer wavelengths occurs from the excited phenolate anion arising from proton dissociation of the OH group of the 6-(p-hydroxyphenyl) substituent of coelenterazine in the direction to His22, which is located within hydrogen bond distance. It is well known that the pK* of a phenolic group is several units below its groundstate pK [54]. If this pK* falls well below 6.5, which is the expected pK of His, rapid transient proton dissociation and its ‘transient displacement’ toward the N atom of His will occur, with simultaneous generation of the excited phenolate anion. As its fluorescence life1439

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A

B

Fig. 7. Two-dimensional drawing of the hydrogen bond network (A), and stereoview of the coelenteramide molecule with the key residues facing into the substrate-binding cavity (B), for Ca2+discharged WT obelin (PDB code 2F8P). Hydrogen bonds are indicated by dashed lines (A) and dots (B). Distances are given in  A. Water molecules are indicated by cyan-colored balls.

time is 5–6 ns [55], there is more than enough time for proton dissociation before radiation. However, in addition to the His residue, the p-OH of the phenol attached at C6 of coelenterazine is also surrounded by Trp and Tyr in aequorin and mitrocomin or Trp and Phe in obelin and clytin within hydrogen bonding distance. All these residues affect the bioluminescence spectrum of photoproteins. Substitution of Trp92 in obelin [42,43] and Trp86 in aequorin [44] to Phe leads to the appearance of a bimodal spectrum, with the kmax at 410 and 470 nm for W92F obelin and 400 and 465 nm for W86F aequorin. For both mutants, the contribution of the violet band corresponding to light emission from the excited coelenteramide in its neutral state (400–410 nm) is almost equal to that at the longer wavelength (465–470 nm). As the crystal structure of W92F obelin [42,43] showed no significant changes in either overall structure or the dimensions of 1440

the substrate-binding cavity compared to WT obelin, the appearance of an intense violet band was suggested to be due to the absence of a hydrogen bond between Ne of Trp and the OH group of the 6-(p-hydroxyphenyl) substituent of coelenterazine. It should be noted that the obelin double mutant W92F/H22E displays a monomodal bioluminescence spectrum with kmax at 390 nm [56]. This suggests that these substitutions hamper dissociation of the proton from the OH group of the 6-(p-hydroxyphenyl) substituent. Most likely, the cause is both lack of a hydrogen bond with Trp92 and different donor/acceptor properties of the side chain of Glu as compared to His. However, without knowledge of the spatial structure of this mutant, we may only suggest possible mechanisms to explain the observed effect. As mentioned above, substitution of Phe88 to Tyr shifts the bioluminescence spectrum of F88Y obelin FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

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(kmax = 453 nm) and the fluorescence spectrum of its Ca2+-discharged state (kmax = 487 nm) toward those of aequorin (kmax for bioluminescence = 469 nm, kmax for fluorescence = 466 nm), although the match is not exact [47]. It was proposed [47] that these changes are the result of formation of an additional hydrogen bond between the hydroxyl group of Tyr and the oxygen atom of the 6-(p-hydroxyphenyl) group of coelenterazine. The spatial structures of F88Y obelin and its Ca2+-discharged conformation state described here support this suggestion, and clearly explain the nascent spectral changes. In aequorin, the hydroxyl group of the 6-(p-hydroxyphenyl) substituent of coelenterazine is positioned at the centre of a triangle formed by the Tyr-Trp-His triad, and forms hydrogen bonds with the phenolic oxygen of Tyr82 and the N atom of His16 (Fig. 5). The Ne of Trp86 may also be hydrogenbonded with the oxygen atom of Tyr82. Although the hydrogen bond between Trp86 and the oxygen of the 6-(p-hydroxyphenyl) group is not detected by the PyMOL program, we cannot exclude its existence, as the  (Fig. 5), and may separation is borderline (3.14 A) well be more evident at room temperature due to protein structural fluctuations. In WT obelin, Phe88 is found in the position corresponding to Tyr82 in aequorin. Consequently, the hydrogen bond network at the oxygen of the 6-(p-hydroxyphenyl) substituent differs from that of aequorin: only a single hydrogen bond with the N atom of His22 has been found (Fig. 4). Similar to aequorin, a hydrogen bond was not detected between Trp92 and the oxygen of the 6(p-hydroxyphenyl) group in WT obelin, although there  for hydrogen is a qualifying Ne–O separation (3.26 A) bond formation (Fig. 4). As substitution of Phe88 by Tyr in obelin leads to the appearance of a hydrogen bond network (Fig. 3) that exactly coincides with that of aequorin (Fig. 5), we may conclude that the additional hydrogen bond between the hydroxyl group of Tyr and the oxygen of the 6-(p-hydroxyphenyl) substituent of coelenterazine in aequorin compared to obelin is responsible for the difference in the bioluminescence spectra of these photoproteins. It is worth noting that, in Ca2+-discharged F88Y obelin, the Tyr88-Trp92His22 triad maintains the same hydrogen bond network as before the reaction (Fig. 6). Although the spatial structure of Ca2+-discharged aequorin is not yet available, we may reasonably assume that its spatial structure will be either very similar or identical to that of Ca2+-discharged F88Y obelin mutant. Despite Ca2+-regulated photoproteins are found in many coelenterates [3], sequencing information is available for only four hydromedusan photoproteins: aequorin, obelin, clytin and mitrocomin (Fig. 1). These FEBS Journal 281 (2014) 1432–1445 ª 2014 FEBS

Structures of F88Y obelin mutant in two states

photoproteins show a high degree of identity of amino acid sequences (65–75%) [7], and the residues forming the internal substrate-binding cavity are strictly conserved (Fig. 1). Despite this, the bioluminescence spectral maxima of recombinant aequorin and mitrocomin are in the range 462–470 nm [8,22,47], whereas those for recombinant obelins and clytin occur at wavelengths of 475–495 nm [15,21]. In addition, in contrast to aequorin and mitrocomin, the bioluminescence spectra of obelin and clytin display a shoulder at 400 nm. Hydromedusan photoproteins themselves are practically non-fluorescent in the visible region until after addition of Ca2+ to produce the bioluminescence reaction, after which their products have efficient fluorescence [8]. It should be pointed out that the fluorescence spectra of Ca2+-discharged aequorin and mitrocomin match their bioluminescence spectra [8,47], whereas for Ca2+-discharged obelin and clytin, the fluorescence is shifted ~ 25–30 nm to longer wavelengths [15,21]. The important difference between these groups of photoproteins is that, in obelin and clytin, Phe is found at sequence positions 88 and 91, respectively, whereas the corresponding position is occupied by Tyr in aequorin and mitrocomin (Fig. 1). Taking into account the structures of two conformation states of the F88Y obelin mutant and a QM/MM (quantum mechanics/molecular mechanics) calculation showing that the light emission wavelength strongly depends on the distance at which the proton is located relative to the oxygen of the 5-(p-hydroxyphenyl) substituent [57], we may reasonably assume that the different arrangement of the hydrogen bond networks results in a different position of the proton towards the N atom of His and the oxygen of the 5-(p-hydroxyphenyl) group of the excited coelenteramide, and therefore to different light emission colors. As the Phe and Tyr residues in clytin and mitrocomin are present at the same positions as in obelin and aequorin, respectively, we may conclude that the mechanism of formation of the longer-wavelength light-emitting coelenteramide excited state in clytin corresponds to that in obelin, whereas the mechanism of formation of the excited state in mitrocomin corresponds to that in aequorin. In summary, in this study, we report the spatial structures of two conformation states of F88Y obelin with bioluminescence spectral properties similar to those of aequorin. A comparison of the hydrogen bond network formed by 2-hydroperoxycoelenterazine and coelenteramide with the key residues facing into the substrate-binding cavity of WT obelin, F88Y obelin mutant, and aequorin clearly shows that the main factor determining the different light emission colors of hydromedusan photoproteins is the different 1441

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Structures of F88Y obelin mutant in two states

arrangement of the hydrogen bond network near the oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine due to the presence of either a Phe or a Tyr residue.

Experimental procedures Preparation of photoprotein samples Site-directed mutagenesis was performed using the Escherichia coli pET19-OL8 expression plasmid carrying O. longissima WT apo-obelin [58] as a template with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions [32]. For apophotoprotein production, transformed E. coli BL21 Gold cells were cultivated with vigorous shaking at 37 °C in LB medium containing ampicillin. Induction was initiated using 1 mM isopropyl thio-b-D-galactoside when the culture reached an attenuance at 590 nm of 0.6–0.8. After addition of isopropyl thio-b-D-galactoside, cultivation of cells was continued for 3 h. The F88Y mutant was purified and activated using coelenterazine as described elsewhere [59–61]. The purified photoprotein was homogeneous based on analysis by SDS/PAGE.

Crystallization, data collection, structure solution and crystallographic refinement For crystallization, the F88Y obelin obtained after ionexchange chromatography was exchanged into a buffer containing 2 mM EDTA, 10 mM Bis-Tris, pH 6.5, and concentrated to ~ 16 mg
mL 1 using Millipore centrifuge tubes (EMD Millipore Corporation, Billerica, MA, USA). The obelin mutant charged with high-purity coelenterazine (JNC Corporation, Yokohama, Japan) was used for these experiments. To prepare Ca2+-discharged F88Y obelin, a solution of the obelin mutant was diluted using 4 mM CaCl2, 10 mM Bis-Tris, pH 6.5, at 4 °C to a final concentration of ~ 1 mg
mL 1. During this procedure, bright blue bioluminescence was observed. After bioluminescence emission ceased, the yellow protein solution had turned colorless, indicating conversion of coelenterazine into coelenteramide. To test the presence of bound coelenteramide, the fluorescence of the final product was measured. The Ca2+-discharged protein was then concentrated to 1 ~ 10 mg
mL using Millipore centrifugal tubes. A Mosquito crystallization robot (TTP LabTech, Melbourn, UK) and commercially available screening kits were used for screening initial crystallization conditions. The hits were optimized manually using the hanging-drop vapor-diffusion technique. The best solution for crystallization of F88Y obelin was 2.1 M D,L-malic acid, pH 7.0 (Emerald BioSystems, Seattle, WA, USA). The F88Y obelin crystals grew as light yellow rod-shaped crystals after 3 days at

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16 °C. It should be noted that F88Y obelin crystals grew only when the drop comprised 2 lL protein and 1 lL precipitant solutions, respectively. The crystal of Ca2+-discharged F88Y obelin was grown by mixing 2 lL protein and 2 lL precipitant solutions during 2 weeks at 4 °C. The best conditions to crystallize Ca2+-discharged F88Y obelin were a solution of 2.2 M ammonium sulfate, 2% polyethylene glycol 400, 0.1 M HEPES-Na, pH 7.5 (Hampton Research, Aliso Viejo, CA, USA). For X-ray analysis, the crystal was directly picked up from the crystallization drop using a fiber loop and flash-frozen in liquid nitrogen. Data were collected at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility, China. Native diffrac tion data were indexed, integrated and scaled to 2.09 A 2+  resolution for F88Y obelin and 1.5 A resolution for Ca discharged F88Y obelin (Table 2) using the HKL2000 software suite [62]. Phases were determined by molecular replacement using PHASER [63], with the structure of WT obelin (PDB code 1QV0) and Ca2+-discharged WT obelin (PDB code 2F8P) as search models. The final models were refined using PHENIX [64] and Refmac5 [65]. Manual adjustments to the model were performed using COOT [66]. RMSD values were calculated using the program Superpose Molecules (CCP4) [67]. Visualization and superposition of the molecular structures as well as detection of hydrogen bonds were performed using PyMOL (Delano Table 2. Data collection and refinement statistics.

Crystal name Data processing Resolution range ( A) Wavelength ( A) Space group Cell dimensions ( A)

Unique reflections Completeness (%) Mean I/r (I) Rmerge (%) Multiplicity Refinement Resolution range ( A) Reflections used (free) Rwork (Rfree) Number of protein atoms Number of ligand atoms Number of solvent atoms Mean B ( A2) RMSD bond lengths ( A) RMSD bond angles (°)

F88Y obelin (PDB code 4N1F)

Ca2+-discharged F88Y obelin (PDB code 4N1G)

50.00–2.09 (2.16–2.09) 0.9789 P41 a = 73.33, b = 73.33, c = 53.08 16 926 (1713) 99.88 (99.12) 22.94 (6.38) 6.7 (37.1) 7.2 (7.4)

50.00–1.50 (1.55–1.50) 0.9789 P212121 a = 60.92, b = 85.13, c = 90.09 58 909 (641) 77.78 (8.61) 25.85 (1.75) 6.3 (44.3) 5.6 (1.1)

43.00–2.09 16906 (1691) 17.16% (19.92%) 1512 34 164 41.80 0.01 1.05

49.54–1.50 58830 (2001) 16.88% (20.89%) 3153 68 566 22.60 0.02 1.73

Values in parentheses are for the highest-resolution shell.

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Scientific, Portland, OR). Parameters to detect hydrogen  for an ideal geometry and 3.2 A  for minbonds were 3.6 A imally acceptable geometry, 180° for a hydrogen bond cone, and 63° for the maximal hydrogen bond angle [68]. Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 4N1F and 4N1G for F88Y obelin and its Ca2+-discharged conformation state, respectively.

Acknowledgements We acknowledge use of beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (China). This work was supported by Russian Foundation for Basic Research grants 12-04-91153 and 12-04-00131, and a China–Russia international collaboration grant from the Chinese Academy of Sciences and the Natural Science Foundation of China, and by the Program of the Government of the Russian Federation ‘Measures to Attract Leading Scientists to Russian Educational Institutions’ (grant 11.G34.31.0058), the ‘Molecular and Cellular Biology’ Program of the Russian Academy of Sciences, and the grant of the President of the Russian Federation ‘Leading Science School’ (3951.2012.4).

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