Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

Michal Nemergut, Daniel Pluskal, Jana Horackova, Tereza Sustrova, Jan Tulis, Tomas Barta, Racha Baatallah, Glwadys Gagnot, Veronika Novakova, Marika Majerova, Karolina Sedlackova, Sérgio M. Marques, Martin Toul, Jiri Damborsky, Zbynek Prokop, David Bednar (), Yves L. Janin () and Martin Marek ()
Additional contact information
Michal Nemergut: Masaryk University
Daniel Pluskal: Masaryk University
Jana Horackova: Masaryk University
Tereza Sustrova: Masaryk University
Jan Tulis: Masaryk University
Tomas Barta: Masaryk University
Racha Baatallah: Institut Pasteur, UMR 3523, CNRS
Glwadys Gagnot: Institut Pasteur, UMR 3523, CNRS
Veronika Novakova: Masaryk University
Marika Majerova: Masaryk University
Karolina Sedlackova: Masaryk University
Sérgio M. Marques: Masaryk University
Martin Toul: Masaryk University
Jiri Damborsky: Masaryk University
Zbynek Prokop: Masaryk University
David Bednar: Masaryk University
Yves L. Janin: Muséum National d’Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université
Martin Marek: Masaryk University

Nature Communications, 2023, vol. 14, issue 1, 1-20

Abstract: Abstract 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 are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. 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 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. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Date: 2023
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DOI: 10.1038/s41467-023-43403-y

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