Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin.

Satoshi Inouye,Mikako Shirouzu,Jumpei Taguchi,Yuto Sumida,Yuri Tomabechi,Takamitsu Hosoya,Parag A Deshpande

doi:10.1371/journal.pone.0251743

Satoshi Inouye, Mikako Shirouzu + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0251743

Copy DOI

Abstract

The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.

Highlights

Coelenterazine with an imidazopyrazinone structure (3,7-dihydroimizazopyrazin-3-one) is widely distributed among luminous and non-luminous marine organisms including coelenterates, arthropods, squids, and fishes [1]
Coelenterazine serves as a light-emitting source of the Ca2+-binding photoproteins (a complex of (S)-2-peroxycoelenterazine and an apoprotein) including aequorin (PDB ID: 1EJ3) [5], obelin (PDB ID: 1QV1) [6] clytin (PDB ID: 3KPX) [7], and mitrocomin (PDB ID: 4NQG) [8], which were identified in the coelenterates
The well-characterized Ca2+-binding photoprotein aequorin emits light by an intramolecular reaction with a trace amount of Ca2+ (>10−7 M), yielding the blue fluorescent protein [BFP, a complex of coelenteramide and apoprotein] and CO2, as shown in the following reaction scheme [9,10,11] (Fig 2): apoAequorin, which consists of 189 amino acid residues [12], can be regenerated to aequorin by incubation with coelenterazine and O2, both in vivo [13,14,15] and in vitro [16, 17] (Fig 3)

Summary

Introduction

Coelenterazine with an imidazopyrazinone structure (3,7-dihydroimizazopyrazin-3-one) is widely distributed among luminous and non-luminous marine organisms including coelenterates (jellyfish and colonial cnidarian), arthropods (copepod, ostracod, and crustacean), squids, and fishes [1]. The oxidation of coelenterazine with O2 by luciferase results in light emission, accompanied by the production of coelenteramide and CO2 according to the following reaction scheme: (Fig 1). The well-characterized Ca2+-binding photoprotein aequorin emits light by an intramolecular reaction with a trace amount of Ca2+ (>10−7 M), yielding the blue fluorescent protein [BFP, a complex of coelenteramide and apoprotein (apoAequorin)] and CO2, as shown in the following reaction scheme [9,10,11] (Fig 2): apoAequorin, which consists of 189 amino acid residues [12], can be regenerated to aequorin by incubation with coelenterazine and O2, both in vivo [13,14,15] and in vitro [16, 17] (Fig 3)

Methods

Results

Conclusion