Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations

Abstract

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca 2؉ -dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd 2؉ -loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156 -Arg-39 -Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramidebinding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.

Thermostability of Ctenophore and Coelenterate Ca2+-Regulated Apo-photoproteins: A Comparative Study

“…The amplified fragment was digested by Sal I and Xho I and then cloned into the pPROEX HTB expression vector. Apo-mnemiopsin (using a pPROEX HTB vector containing apo-mnemiopsin gene, GenBank entry GQ231544 ) and apo-aequorin were expressed in Escherichia coli BL21 (DE3). Thus, strains were cultivated in Lysogeny broth (LB) medium at 37 °C and subjected to reciprocal shaking (250 rpm) for 12–14 h. Then, 100 mL of medium with 1 mL of the precultured cell was inoculated and grown at 37 °C until the OD 600 reached ∼0.5–0.6, and protein expression was induced with 1 mM IPTG.…”

“…Crystal structures of some members of the coelenterate photoprotein family have been characterized, including those of aequorin, obelin, and clytin, in different states of calcium-, coelenterazine-, ,− and coelenteramide ,, -bound states. From the ctenophore photoprotein family, the only published crystal structures are apo-mnemiopsin1 and apo-berovin. , Light emission occurs via a similar reaction in families of coelenterate and ctenophore.…”

“…As established in several studies, protein pairs with a sequence identity of 20–35% are often termed the “twilight zone” and structural similarity between them is considerably less common . Interestingly, the structural studies indicate that despite a low degree of sequence identity between coelenterate and ctenophore photoproteins, ,, they have high degree of structural similarity. For instance, the sequence identity between mnemiopsin and aequorin is 26.4%, in which two dissimilar sequences appear to be structural homologues.…”

“…For instance, the sequence identity between mnemiopsin and aequorin is 26.4%, in which two dissimilar sequences appear to be structural homologues. To date, numerous studies have been carried out on coelenterate and ctenophore families to identify the amino acid residues involved in the chromophore-binding site around coelenterazine, the mechanism of action, and light emission, ,,,− but the thermostability of photoproteins and the mechanism responsible for it remain unknown.…”

Unusual shift in the visible absorption spectrum of an active ctenophore photoprotein elucidated by time-dependent density functional theory

“…At the same time, similar to hydromedusan photoproteins, ctenophore proteins also contain three canonical EFhand Ca 2+ -binding sites [41]. The structure of any ctenophore photoprotein bound to oxygenated coelenterazine has not yet been determined, and only the tertiary structures of apo-berovin bound with Ca 2+ or Mg 2+ ions [43,44] and apo-mnemiopsin loaded by Cd 2+ ions [45] are available now.…”