Abstracts
Structure, function of luciferases and photoproteins
Atomistic views and proposed mechanism of NanoLuc luciferase actionMartin Marek1, Michal Nemergut2, Daniel Pluskal3, Jana Horackova3, Tereza Sustrova3, Tomas Barta4, Martin Toul2, Zbynek Prokop2, David Bednar2, Yves L. Janin5, Jiri Damborsky2
1Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic & International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic, 2Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno & International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic, 3Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, 4Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic, 5Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France
E-mail: martin.marek@recetox.muni.cz
The widely used NanoLuc luciferase was engineered over ten years ago but the oxidative mechanism by which it generates blue photons remained unclear [1]. Here we decipher NanoLuc luciferase action through crystallographic, spectroscopic, and computational experiments. We show that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface [2]. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site by mutagenesis can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. Thus, we reveal that NanoLuc, despite its structural dissimilarity, employs an analogous catalytic principle to generate blue photons, as we recently revealed for coelenterazine-powered Renilla-type luciferases [3]. References [1] Hall M.P., Unch J., Binkowski B.F., Valley M.P., Butler B.L., Wood M.G., Otto P., Zimmerman K., Vidugirus G., Machleidt T., Robers M.B., Benink H.A., Eggers C.T., Slater M.R., Meisenheimer P.L., Klaubert D.H., Fan F., Encell L.P., Wood K.V. (2012) ACS Chemical Biology 7 (11): 1848-1857.[2] Nemergut M., Pluskal D., Horackova J., Sustrova T., Tulis J., Barta T., Baatallah R., Gagnot G., Novakova V., Majerova M., Sedlackova K., Marques S.M., Toul M., Damborsky J., Prokop Z., Bednar D., Janin Y.L., Marek M. (2023) Nature Communications, 14 (1), art. no. 7864.[3] Schenkmayerova, A., Toul, M., Pluskal, D., Baatallah, R., Gagnot, G., Pinto, G. P., Santana, V. T., Stuchla, M., Neugebauer, P., Chaiyen, P., Damborsky, J., Bednar, D., Janin, Y. L., Prokop, Z., Marek, M. (2023) Nature Catalysis 6: 23-38.
Keywords: Bioluminescence; Oplophorus gracilirostris; luciferase; coelenterazine; catalysis; allostery; azacoelenterazine
Acknowledgments: The authors would like to express their thanks to the Czech Science Foundation (GA22-09853S). D. P. is a Brno Ph.D. Talent Scholarship holder funded by the Brno City Municipality.