Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca 2؉ -regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously 15 N, 13 C, 2 H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain 1 H-15 N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K D ؍ 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.The bioluminescence of many marine coelenterates, wellstudied examples being the jellyfish Aequorea and the seapansy Renilla, involves the interaction of two proteins, a Ca 2ϩ -regulated photoprotein in the jellyfish case, aequorin, and its cognate green-fluorescent protein, Aequorea GFP (1). Addition of Ca 2ϩ to the purified aequorin produces a blue bioluminescence. It was early recognized that, in the jellyfish itself, the in vivo bioluminescence was a green color and after further study, the origin of this green emission was identified as the GFP. A Förster-type resonance energy transfer (FRET) 3 mechanism was invoked to explain how this bioluminescence spectrum is shifted (2). However, the well-known Förster theory requires concentrations of the donor-acceptor partners in the millimolar range, whereas in some bioluminescence systems, e.g. from the sea pansy Renilla and also the jellyfish Clytia subject herein, the GFP effect on the in vitro bioluminescence is observed at micromolar concentrations (3). Clearly the bioluminescence interaction has to involve formation of a complex and, in the case of Renilla, the formation of a luciferase-GFP complex has been shown (3).In this work, we have determined by x-ray crystallography the spatial structures of th...
The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.
In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca 2+ -regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.
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