Crystal structure of obelin after Ca²⁺-triggered bioluminescence suggests neutral coelenteramide as the primary excited state

Abstract

The crystal structure at 1.93-Å resolution is determined for the Ca 2؉ -discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound Ca 2؉ and͞or coelenteramide. Among these structures, global conformational changes are small, typical of the class of ''calcium signal modulators'' within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.coelenterazine ͉ photoprotein ͉ EF hand ͉ luciferase ͉ aequorin

Expression and characterization of the calcium-activated photoprotein from the ctenophore Bathocyroe fosteri: Insights into light-sensitive photoproteins

“…Interestingly, previous mutagenesis experiments targeting the obelin (cnidarian) active site have identified key residues involved in coelenterazine binding that are substituted in ctenophores [26,27]. Some of the residues involved in stabilizing coelenterazine in obelin (Tyr138, His175, Trp179, Tyr190) and in generating or stabilizing the excited state (His22, Trp92) [26] are non-conservatively replaced in BfosPP.…”

“…Some of the residues involved in stabilizing coelenterazine in obelin (Tyr138, His175, Trp179, Tyr190) and in generating or stabilizing the excited state (His22, Trp92) [26] are non-conservatively replaced in BfosPP. Most notably His175 is replaced by the non-hydrogen-bonding phenylalanine (Table 1).…”

“…When previous mutagenesis experiments in aequorin replaced the same histidine residue with alanine, phenylalnine or tryptophan, all luminescence activity was lost [28]. Thus replacement of this histidine in ctenophore photoproteins might be partly responsible for photoinactivation, since there is no residue available for hydrogen bonding with Tyr202 (numbered according to BfosPP) which stabilizes the 2-hydroperoxy group of bound coelenterazine, where decarboxylation occurs [26]. Even though previous experiments with aequorin reported loss of activity upon substitution of histidine, it is possible these experiments were not performed in the dark, and therefore loss of activity may have been due to subsequent photoinactivation.…”

Protein-protein complexation in bioluminescence

“…This disturbs the hydrogen bond network around the peroxy group, which in turn triggers decarboxylation of the 2-hydroperoxycoelenterazine, ending up in formation of the product excited coelenteramide and emission of blue light ( Fig. 2D) (Head et al, 2000;Liu et al, 2006). Clytin completes only one turnover in vitro because after the bioluminescence reaction the product remains tightly bound.…”

Coelenterazine-dependent luciferases

“…Binding of Ca 2+ causes small conformational changes in a substrate binding cavity of the protein, which disturb the hydrogen bond network that stabilizes 2 hydroperoxycoelenterazine, thereby triggering the reaction of oxidative decarboxylation leading to the formation of the product in the excited state [31]. Unlike Ca 2+ regulated photoproteins, the photoproteins of squid Symplectoteuthis oualaniensis [15] and mollusc Pholas dactylus [16], simplectin and pholasin, are a complex of a protein with a covalently bound coelenterazine, which is formed upon binding of dehydrocoelenterazine with the apoprotein.…”