Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins

Abstract

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.

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

“…The internal cavity of hydromedusan Ca 2+ ‐regulated photoproteins which also use coelenterazine as a substrate of bioluminescence reaction contains many aromatic residues . These residues are involved in stabilizing the 2‐hydroperoxy group of coelenterazine (Trp, His and Tyr) , emitter formation (His, Tyr and Trp) and coelenterazine activation by oxygen (Tyr) . In copepod luciferases, conservative motifs within the repeats contain two Tyr residues one of which is conservative (positions are marked with frames in Fig.…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…Although aequorin structure has been the most extensively studied by mutagenesis, several reports by Vysotski and collaborators have also shown striking structure–function effects in obelin . In 2008, Rowe et al .…”

“…Although aequorin structure has been the most extensively studied by mutagenesis, several reports by Vysotski and collaborators have also shown striking structure-function effects in obelin (87,95,98,99). In 2008, Rowe et al managed to produce aequorin and obelin variants with new emission colors and altered decay kinetics, by combining mutations with synthetic coelenterazines (100).…”

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

“…1); that is, it resembles the case of aequorin and obelin-whereas Tyr is found near OH group of the 6-(p-hydroxyphenyl) substituent of coelenterazine in aequorin, in obelin the corresponding position is occupied by Phe (27,29). And this distinction accounts for the difference in the emission spectra of these photoproteins (27,29).…”