NMR-derived Topology of a GFP-photoprotein Energy Transfer Complex

Abstract

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...

Perspectives on Bioluminescence Mechanisms

“…The effect is less with obelin and Clytia GFP, suggesting specificity in the interaction, as also observed with the above mentioned other cases . The same argument used previously was that the spectral effect at these concentrations of Clytia GFP implied the presence of a protein–protein complex with K D in the μ m range but a search using standard methods, analytical ultracentrifugation, size‐exclusion chromatography, surface plasmon resonance or polarization fluorometry, failed to detect any complex with a K D < 100 μ m .…”

Protein-protein complexation in bioluminescence

“…This apparently minimizes energy dissipation upon fluorescence excitation to favor a high quantum yield of the Clytia GFP fluorescence (0.86; maximum 500 nm upon excitation at 470 nm) . From analytical ultracentrifugation, size-exclusion chromatography and fluorescence anisotropy data, Clytia GFP is an obligate dimer (Titushin et al, 2010;Malikova et al, 2011). The dimerization interface of the Clytia GFP monomer reveals hydrogen bonding and an accessible surface area value (1370 Å 2 ) favorable for strong dimerization (Jones and Thornton, 1996).…”

“…As in the case of bacterial luciferase and lumazine protein, the energy transfer in the Clytia GFP-clytin system is rationalized by the FRET mechanism. The requirement of donor-acceptor separation to be less than 100 Å at micromolar concentrations of clytin and Clytia GFP can only be achieved assuming a tight protein-protein interaction (Förster, 1960;Wu and Brand, 1994;Titushin et al, 2010;Markova et al, 2010). Although the bioluminescence spectrum shift is complete at micromolar amounts of proteins, implying K D of Clytia GFP-clytin complexation to be in the micromolar range, no evidence for protein-protein association could be found from analytical ultracentrifugation, size-exclusion chromatography, surfaceplasmon resonance, or polarization fluorometry experiments, which are capable of detecting complexation with a K D below 10 -4 mol/L (Titushin et al, 2010;Malikova et al, 2011 (Fig.…”

“…Several lines of evidence indicate that delivery of luciferin from the binding protein to the luciferase as well as modulation of bioluminescence spectra as a result of energy transfer to the antenna proteins, involves protein-protein interactions (Morise et al, 1974;Ward andCormier, 1976, 1978;Nicolas et al, 1991;Cutler, 1995;Schultz et al, 2005;Titushin et al, 2008Titushin et al, , 2010Stepanyuk et al, 2009;. The interactions are of a transient nature, with K D in the range of 10 −6 -10 −3 mol/L, which highly impedes direct detection of the complexes, or their structural studies with Xray crystallography or NMR methods.…”

“…For those bioluminescent systems where crystal structures of the individual proteins are known, the topologies of the protein-protein complexes were solved by means of data-driven computer modeling. Among the structures available so far, there are those of the lumazine protein-luciferase complex from the bacteria Photobacterium (Sato et al, 2010), the GFPphotoprotein clytin complex from the jellyfish Clytia (Titushin et al, 2010), and the coelenterazine-binding protein-luciferase complex from the soft coral Renilla (Stepanyuk et al, 2009). The main goal of this review is to cover the mentioned bioluminescence systems, where extensive biochemical studies of the protein-protein interactions have been complemented with structural data, resulting in new insight into the bioluminescence mechanism with regard to the role of the protein-protein complexes.…”