Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction
Abstract:Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F o… Show more
“…The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild‐type obelin gave clear evidence that substitution of Tyr by Phe had no effect on the overall structure of both active and Ca 2+ ‐discharged Y138F obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild‐type obelins, there was a small but substantial difference—in the coelenterazine‐binding cavity of Y138F obelin, there was no water molecule like one found in the wild‐type obelin . However, in the Ca 2+ ‐discharged Y138F obelin this water molecule appeared in the expected location.…”
Section: Tyr Residuesmentioning
confidence: 93%
“…However, in the Ca 2+ ‐discharged Y138F obelin this water molecule appeared in the expected location. It was proposed that this result, along with much slower bioluminescence kinetics of Y138F obelin , points to the involvement of this water molecule in the decarboxylation reaction of the 2‐hydroperoxycoelenterazine by means of protonation of a dioxetanone anion before its decomposition into the excited‐state product .…”
Section: Tyr Residuesmentioning
confidence: 99%
“…The reason behind the loss of bioluminescence activity by aequorin at substitution of His58 to Phe is not so clear. However, the determination of spatial structures of Ca 2+ ‐discharged wild‐type obelin and its Y138F mutant in two conformational states has led to the plausible suggestion of the role of this conserved histidine in photoprotein bioluminescence. It was proposed that His in this position together with other residues and a water molecule might participate in proton transfer from solvent to the dioxetanone anion .…”
Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.
“…The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild‐type obelin gave clear evidence that substitution of Tyr by Phe had no effect on the overall structure of both active and Ca 2+ ‐discharged Y138F obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild‐type obelins, there was a small but substantial difference—in the coelenterazine‐binding cavity of Y138F obelin, there was no water molecule like one found in the wild‐type obelin . However, in the Ca 2+ ‐discharged Y138F obelin this water molecule appeared in the expected location.…”
Section: Tyr Residuesmentioning
confidence: 93%
“…However, in the Ca 2+ ‐discharged Y138F obelin this water molecule appeared in the expected location. It was proposed that this result, along with much slower bioluminescence kinetics of Y138F obelin , points to the involvement of this water molecule in the decarboxylation reaction of the 2‐hydroperoxycoelenterazine by means of protonation of a dioxetanone anion before its decomposition into the excited‐state product .…”
Section: Tyr Residuesmentioning
confidence: 99%
“…The reason behind the loss of bioluminescence activity by aequorin at substitution of His58 to Phe is not so clear. However, the determination of spatial structures of Ca 2+ ‐discharged wild‐type obelin and its Y138F mutant in two conformational states has led to the plausible suggestion of the role of this conserved histidine in photoprotein bioluminescence. It was proposed that His in this position together with other residues and a water molecule might participate in proton transfer from solvent to the dioxetanone anion .…”
Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.
“…W 1 and W 2 are protein‐bound water molecules. (Adapted from reference , and reproduced with permission of the International Union of Crystallography).…”
The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns-ps) fluorescence spectroscopy, NMR, X-ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high-energy species that undergoes decarboxylation to form directly the product in its S 1 state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ "antenna proteins," lumazine protein and the greenfluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca 2+ -regulated photoproteins and the antenna protein interactions.
“…In the past decade owing to determination of spatial structures of ligand‐dependent conformational photoprotein states , spatial structures of photoprotein variants and comprehensive mutagenesis studies , significant progress has been attained in understanding the function of certain active site residues in oxidative decarboxylation of 2‐hydroperoxycoelenterazine and emitter formation. At the same time, the reason why hydromedusan photoproteins revealing a high degree of sequence and structural identity display different bioluminescent kinetics and sensitivity to calcium still remains an enigma.…”
Upon binding their metal ion cofactors, Ca -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca -regulated photoproteins.
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