Fig. 6: Molecular docking and computational simulations of NanoLuc complexes.

a Visualization of the best poses of FMZ (magenta sticks) from molecular docking to the NanoLuc monomer dimer (chain A cyan and chain B blue cartoon), in comparison with the crystal-bound FMA (yellow sticks). b Two potential access tunnels (yellow and blue spheres) to the central luciferin binding cavity (green spheres) of NanoLucCTZ (salmon cartoon) are shown from the front and the side view. The tunnels were computed on Caver Web⁸⁴. c The final poses of CTZ (orange sticks) from ASMD simulations of NanoLuc structures in the closed and open β-barrel state, and NanoLuc^CTZ open β-barrel structure. The crystal-bound azaCTZ (transparent green sticks) is shown in each picture for reference. The interacting aromatic residues are shown as thin sticks. The distances (in Å) between H2/H3 and H3/H4 helices are indicated by yellow dashed lines. Associated MD trajectories are shown in Supplementary Movies 1–3. d NanoLuc closed β-barrel structure with shown mutations (L27V, K33N, K43R, and Y68D) introduced during the last third phase of NanoLuc development responsible for maximizing FMZ fluorescence. e Equilibrium probabilities of FMZ macrostates bound in the surface pocket of monomeric and dimeric NanoLuc. The calculation was bootstrapped 100 times using a random 80% of the data. The height of the bars corresponds to the mean value and the error bars show the standard deviation. f The potential of mean force needed to steer the two subunits of different NanoLuc dimers up to 12 Å apart, obtained by adaptive steered molecular dynamics.