Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein

Abstract

Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca²⁺-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O₂ only subsequent to Ca²⁺ binding. CBP belongs to the EF-hand superfamily of Ca²⁺-binding proteins and contains three “EF-hand” Ca²⁺-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 Å, is found to approximate the protein scaffold characteristic of the class of Ca²⁺-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca²⁺. Addition of Ca²⁺ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.