Fig. 5: Structural characterization of coelenteramide binding to the catalytically defective RLuc8-W121F/E144Q mutant.

a Cartoon representation of the overall structure of the RLuc8-W121F/E144Q mutant with coelenteramide (CEI) in its active site. CEI is the product of the LUC reaction and shown as cyan space-filling spheres. Residues of the conserved catalytic pentad are shown as purple spheres; the central eight-stranded β-sheet is coloured yellow; the α4 helix and L9 loop (L9-α4 element) are coloured violet, and the L14 loop is coloured orange. b Cutaway surface representation of the enzyme active site cavity with the bound CEI (shown as cyan sticks). The colouring is the same as in panel (a). Water molecules are shown as red spheres. c Close-up view of structural superposition of RLuc8-W121F/E144Q (green), RLuc8 (PDB ID 2PSF A; cyan) and RLuc8 (PDB ID 2PSF B; teal). CEI is shown as cyan space-filling spheres. Note the conformational sampling of the L9-α4 fragment. The bottom 4-hydroxyphenyl group connected to the CEI acetamide moiety is deeply buried in the active site cleft, where it is anchored in the slot tunnel through multiple hydrophobic (P224, I223 and I266) and aromatic π-stacking (W156) interactions. The 4-hydroxyphenyl group interacts with the indole NH group of W156 through a water-mediated hydrogen bond bridge. The acetamide moiety of CEI is positioned close to the conserved catalytic centre. In chain B, the top 4-hydroxyphenyl group linked with the CEI pyrazine ring interacts with a side chain of K189 through a water-mediated hydrogen bond bridge and forms a hydrogen bond with the carboxylate group of D162 from a symmetry-related enzyme molecule.