Journal of Molecular Biology
Volume 374, Issue 4, 7 December 2007, Pages 1017-1028
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Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis

https://doi.org/10.1016/j.jmb.2007.09.078Get rights and content

Abstract

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

Introduction

Luciferases have become important research tools over the last two decades due to their ability to emit light and therefore be monitored externally to the milieu they reside in. This ability has seen these bioluminescent proteins being utilized widely as reporter genes in cell culture experiments and, more recently, in the context of small animal imaging.1 The two main classes of luciferases employed as research tools are the beetle and coelenterazine luciferases. The beetle luciferases (e.g., firefly) use d-luciferin as their substrate, are highly similar (≥45%),2 and have been extensively studied, including structurally.3

In contrast, no similarity has been seen among most of the coelenterazine-using luciferases identified so far (e.g., Gaussia, Renilla, Pleuromamma, and Oplophorus),4 even when the luciferases originate from species within the same family (Gaussia versus Pleuromamma). This has been taken to indicate that coelenterazine-utilizing luciferases have emerged multiple times throughout the course of evolution. Of the coelenterazine luciferases, the luciferase from Renilla reniformis (RLuc) has been the most extensively studied5 and the most widely employed for research. To date, however, no structure has been elucidated of any of the coelenterazine-using luciferases, including RLuc.

In R. reniformis, RLuc is found in membrane-bound intracellular structures within specialized light-emitting cells,6,7 along with two other proteins, a closely interacting green fluorescent protein (RrGFP)8 and a Ca2+-activated luciferin-binding protein9 (the almost identical luciferin-binding protein from Renilla mülleri has recently been crystallized10). The chemical reaction that transpires in RLuc-mediated bioluminescence involves catalytic degradation of coelenterazine and proceeds through a dioxetane (also called dioxetanone or cyclic peroxide) intermediate step.11 In vitro, the reaction yields blue light (480-nm peak); however, in vivo, RrGFP, not RLuc, is the light emitter. The energy released by the luciferase-catalyzed oxidation of coelenterazine is passed via resonance energy transfer to the fluorophore of RrGFP and emitted as a green-wavelength photon,12 explaining the 505-nm peaked bioluminescence observed from the animal.

Since the cloning of the gene for RLuc,13 this luciferase has been widely used in molecular biology, mainly as a reporter gene. More recently, the gene has been incorporated into reporter applications of increasing complexity, including fusion reporter genes,14, 15, 16 split reporter complementation systems,17 and resonance energy transfer-based sensors.18,19 Work has also emerged utilizing variants of Renilla luciferase to create novel imaging probes by fusing the luciferase to engineered antibodies20 and to generate self-illuminating quantum dots by attaching the luciferase as an internal light source21. In the development of these and other applications, the tertiary structure of the protein would be helpful in understanding the most effective way of employing the luciferase. For instance, structural data would allow assessment of potential steric hindrance issues prior to the creation of fusion protein constructs involving RLuc and which residues are available for site-specific conjugation reactions. Structural data would also be helpful in guiding rational alteration of the enzyme in pursuit of beneficial modification of its spectral and enzymatic properties.22,23

The current work focused on an 8-mutation variant of RLuc (RLuc8) in lieu of the native enzyme as this variant is more stable and more easily expressed in comparison to the native enzyme.22 The protein's structure was successfully determined from crystals grown under a number of conditions, with a product-bound structure highlighting residues that had previously been found important for determining the enzyme's emission spectrum.23 As RrGFP is known to physically associate with RLuc in vitro, work with RrGFP was pursued as well. RrGFP was found to be structurally very similar to the GFP from Aequorea victoria (AvGFP), with the exception of containing a much stronger dimerization interface.

Section snippets

Protein characterization

Periplasmically expressed and purified RLuc8 was characterized by light scattering and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to assess the oligomeric state and mass of the protein. Light-scattering results indicated that RLuc8 existed as a monomer in solution, as molar-mass-moment calculations indicated a molecular weight of 33.8 kDa (error, 7%) with a relatively low polydispersity across the gel filtration elution profile (∼11%). By mass spectrometry, the

Structures of RLuc8

The structure derived from the RLuc8:diammonium condition is presented in Fig. 1a, with statistics for this and other structures discussed later given in Table 2. Residues 4–308 (of 311 total) were successfully identified in the electron density data. Not identified were 2 residues from the N-terminus, along with 3 residues on the C-terminus and the 6×His tag. The resultant structure from RLuc8:KSCN was almost identical with that from RLuc8:diammonium (Cα RMSD, 0.2 Å), with the sole exception

Discussion

Much like the similar bacterial haloalkane dehalogenases,32 Renilla luciferase has a characteristic α/β-hydrolase fold sequence at its core33 and shares the conserved catalytic triad of residues employed by the dehalogenases.22 The level of primary sequence similarity is somewhat surprising given that the dehalogenases are hydrolases, whereas the luciferase is an oxidase, and hypotheses on how this situation may have come to be from an evolutionary standpoint have previously been discussed.22

Constructs

The plasmids pBAD-pelB-RLuc8 and pBAD-RLuc8, used for periplasmic expression and cytoplasmic expression, respectively, have previously been described22 (rluc8 GenBank identifier 127951035). The proteins expressed from these plasmids contain a noncleavable C-terminal 6×His tag, with the only difference between the two being that the mature protein from the periplasmic construct lacks an N-terminal methionine. An additional periplasmic expression plasmid, pBAD-pelB-6×His-thr-S3RLuc8, containing a

Acknowledgements

This work was supported in part by a Stanford Bio-X Graduate Fellowship (AML), NCI CCNE U54 (SSG), NCI CA114747 ICMIC P50 (SSG), and NCI R01 CA082214 (SSG).

The authors would like to thank Dr. Axel T. Brunger for his critical reading of and comments on the manuscript, as well as for allowing use of his crystallography equipment.

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    A.M.L. and T.D.F. contributed equally to this work

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