Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data

Although the crystal structure of Vibrio hareyi luciferase has been elucida ted, the binding sites for the flavin mononucleotide and fatty aldehyde sub strates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the ex subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-acti vity data for the length of the linker connecting the phosphate group to th e isoalloxazine ring represent important characteristics of the luciferase- bound conformation of the flavin mononucleotide. A model of the luciferase- flavin complex is developed here using flexible docking supplemented by the se structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation searc h by using the Monte Carlo minimization approach. The resulting databases o f energy-ranked feasible conformations of the luciferase complexes with fla vin mononucleotide, omega -phosphopentylflavin, omega -phosphobutylflavin. and omega -phosphopropylflavin were filtered according to the structure-act ivity profile of these analogs. A unique model was sought not only on energ etic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the l uciferase-binding site, an orientation that is also inaccessible to the ina ctive flavin analog. The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature, Specifically, the isoalloxazine ring of the flavin monon ucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the a subunit of luciferase. The model of the bi nary complex reveals a distinct cavity suitable for aldehyde binding adjace nt to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site.