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

Maryam Molakarimi,Michael A Gorman,Ammar Mohseni,Zaiddodine Pashandi,Majid Taghdir,Hossein Naderi-Manesh,Reza H Sajedi,Michael W Parker

doi:10.1074/jbc.ra118.006053

Abstract

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-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 Cd2+-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 coelenteramide-binding 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.

Highlights

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2؉-dependent photoproteins
Based on the structure of aequorin bound to coelenterazine, the proposed substratebinding site of mnemiopsin1 is located in the core of the protein, surrounded by four EF-hand loops that provide a fairly hydrophobic pocket that contains mostly hydrophobic side chains but some hydrophilic aromatic residues
The mechanism of reaction initiation and light emission of the ctenophore family was largely unknown until our recent quantum mechanics/molecular mechanics (QM/MM) studies on berovin, in which we proposed a catalytic mechanism predicting that Asp-158 –Arg-41–Tyr-204 triad, equivalent to the His-175–Tyr-190 –Trp-179 triad in the cnidarian family, could shuttle a proton from the 2-hydroperoxy group of coelenterazine to bulk solvent [25]

Summary

Overall structure

Apo-mnemiopsin has a two-domain fold with four ␣-helices A–D and E–H in the N- and C-terminal domains, respectively (Fig. 1). The loops of EF-hand III and IV interact with each other through hydrogen bonds between main-chain atoms of Val-143 with Leu-177. The presence of three Cd2ϩ loaded EF-hand Ca2ϩ-binding loops is observed in the apo-mnemiopsin structure (Fig. 3). The results showed that coelenterazine docks into the mnemiopsin and berovin structures with a similar binding mode. In the apo-mnemiopsin structure the Tyr-131 hydroxyl group forms a hydrogen bond to a water molecule (Fig. S2), both of which are far from the N1 atom (Fig. 6A). In the QM/MM calculations of Ca2ϩ-discharged mnemiopsin, this water molecule was manually protonated to form a hydronium ion. This water molecule forms a strong hydrogen bond with the amide N atom of coelenteramide (Fig. 6B). Selected bond lengths (Å) of coelenterazine obtained from the MD simulation and QM/MM studies

Discussion

Bacterial strain and growth condition

Initial structure preparation and docking studies

Space group

Molecular dynamics simulation