3KPX

Crystal Structure Analysis of photoprotein clytin

Summary for 3KPX

• Entry DOI: 10.2210/pdb3kpx/pdb
• Descriptor: Apophotoprotein clytin-3, C2-HYDROPEROXY-COELENTERAZINE, CALCIUM ION, ... (4 entities in total)
• Functional Keywords: photoprotein clytin, fluorescent protein, hydrolase
• Biological source: Clytia gregaria (Phialidium gregarium)
• Total number of polymer chains: 1
• Total formula weight: 22951.70

Authors

Titushin, M.S.,Li, Y.,Stepanyuk, G.A.,Wang, B.-C.,Lee, J.,Vysotski, E.S.,Liu, Z.-J. (deposition date: 2009-11-17, release date: 2010-10-06, Last modification date: 2023-11-01)

Primary citation

Titushin, M.S.,Feng, Y.,Stepanyuk, G.A.,Li, Y.,Markova, S.V.,Golz, S.,Wang, B.-C.,Lee, J.,Wang, J.,Vysotski, E.S.,Liu, Z.-J.
NMR derived topology of a GFP-photoprotein energy transfer complex
J.Biol.Chem., 285:40891-40900, 2010

PubMed 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.

PubMed: 20926380
DOI: 10.1074/jbc.M110.133843

Experimental method

X-RAY DIFFRACTION (1.899 Å)