Illuminating the mechanism and allosteric behavior of NanoLuc luciferase
- Nemergut, Michal1
- Pluskal, Daniel2
- Horackova, Jana2
- Sustrova, Tereza2
- Tulis, Jan2
- Barta, Tomas3
- Baatallah, Racha4
- Gagnot, Glwadys5
- Novakova, Veronika1
- Majerova, Marika1
- Sedlackova, Karolina1
- Marques, Sergio M.1
- Toul, Martin1
- Damborsky, Jiri1
- Bednar, David1
- Prokop, Zbynek1
- Janin, Yves L.6
- Marek, Martin1
- 1. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, Bld. C13, 625 00 Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic
- 2. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, Bld. C13, 625 00 Brno, Czech Republic
- 3. Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic
- 4. Unité de Chimie et Biocatalyse, Institut Pasteur, UMR 3523, CNRS, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
- 5. Unité de Chimie et Biocatalyse, Institut Pasteur, UMR 3523, CNRS, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; Université de Paris, 12 rue de l'école de Médecine, 75006 Paris, France
- 6. Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France
Description
NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis
remains puzzling. Here experimental and computational techniques were combined, revealing that
imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on
the enzyme surface. 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 can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel
arginine coordinates the imidazopyrazinone component of luciferin which then react with O 2 via a radical
charge-transfer mechanism, and it also protonates the resulting excited amide product to form a light-
emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, is fine-tuning the blue
color emitter to secure a high emission intensity. This information is critical to engineering the next-
generation of ultrasensitive bioluminescent reporters.
Files
MechanismAllosteryNanoLuc.zip
Additional details
Funding
- European Commission
- ADDIT-CE - Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe 101087124
- European Commission
- SinFonia - Synthetic biology-guided engineering of Pseudomonas putida for biofluorination 814418
- European Commission
- CETOCOEN Excellence - CETOCOEN Excellence 857560