Crystal structure of semisynthetic obelin-v

Abstract

Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca²⁺ -regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca²⁺ -regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.80 Å resolution. The structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen-bond pattern and slightly increased distances between key active site residues and some atoms of 2-hydroperoxycoelenterazine-v were found. The fluorescence quantum yield (Φ_FL ) of obelin bound with coelenteramide-v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme-substrate complexes containing the 2-hydroperoxy adduct of CTZ-v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (Φ_R ) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin-v is caused by the low efficiency of generating an electronic excited state (Φ_S ). In turn, the low Φ_S value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin-v active site.

Keywords: analog; bioluminescence; coelenterazine; coelenterazine-v; obelin; photoprotein; protein structure.

FIGURE 1

Coelenterazine (a) and coelenterazine‐v (b)

FIGURE 2

Crystal structure of obelin‐v. (a) Overall structure of obelin‐v. The helices are marked by capital letters A–H. The loops are designated I–IV. (b) Superimposition of obelin‐v (pink, PDB: 7O3U), obelin bound with one Ca²⁺ (cyan, PDB: 1QV1), and obelin with no Ca²⁺ (green, PDB: 1QV0). The 2‐hydroperoxyCTZ and 2‐hydroperoxyCTZ‐v molecules are displayed as stick models in the center of the protein; calcium ions are shown as balls. The 2‐hydroperoxyCTZs and calcium ions are colored according to the structure color. (c) Electron‐density map of 2‐hydroperoxyCTZ‐v molecule in the substrate binding cavity of obelin‐v. (d) Amino acid sequence of obelin from O. longissima. The helices are shown as pink sticks; the loops involved in the binding of Ca²⁺ are shown as blue sticks

FIGURE 3

Stereoview of the first Ca²⁺‐binding loop of obelin‐v. Calcium ion (green) and water molecules (red) are shown as balls. Oxygen and nitrogen atoms of amino acid residues are colored in red and blue, respectively. Hydrogen bond distances are shown with dashed lines. Distances are indicated in Å

FIGURE 4

Hydrogen bond network formed by Arg21 located in helix A and C‐terminal Pro 195 together with Asp187 and Phe178, which ensures solvent inaccessibility of the substrate‐binding cavity in obelin‐v (a) and obelin (b)

FIGURE 5

Substrate‐binding cavities of obelin‐v and obelin. (a) Stereoview of superimposition of the corresponding 2‐hydroperoxyCTZ molecules with the key residues facing into the substrate‐binding cavities of obelin (cyan) and obelin‐v (pink). The 2‐hydroperoxyCTZs and water molecules are colored according to the structure color, water molecules are shown as balls. Two‐dimensional drawing of the hydrogen bond network in obelin‐v (b) and obelin (c). Hydrogen bonds are shown as dashed lines, possible hydrogen bonds are shown as arrows. Distances are given in Å

FIGURE 6

Absorption spectra. (a) Free CTZ‐v (black), obelin‐v (red) and Ca²⁺‐discharged obelin‐v (blue). (b) Free CTZ (black), obelin (red) and Ca²⁺‐discharged obelin (blue). Left ordinate axes are for absorbance in the range of 250–400 nm, right ordinate axes—for the range of 400–550 nm. CTZs are in ethanol; obelins—in 1 mM EDTA, 20 mM Tris–HCl pH 7.2; Ca²⁺‐discharged obelins—in 1 mM CaCl₂, 20 mM Tris–HCl pH 7.2. All samples are in concentration of 8.93 μM

FIGURE 7

Spectral properties of obelin and obelin‐v. (a) Bioluminescence spectra of obelin (left ordinate, black line) and obelin‐v (right ordinate, red line) normalized to protein concentration. Insertion shows normalized bioluminescence spectra of obelin (black line) and obelin‐v (red line) (b) Fluorescence spectra of corresponding Ca²⁺‐discharged photoproteins divided by the absorption at the wavelength of excitation (373 nm). Vertical lines indicate the wavelength of maxima of the components determined by fitting with the sum of two Gaussian functions