Interplay among the flipping glutamine, a conserved phenylalanine, water and hydrogen bonds within a blue-light sensing LOV domain

In this work we exploited time-resolved photoacoustics (PA) and molecular dynamics (MD) simulations to investigate the function of a conserved phenylalanine residue in blue sensing (BL) LOV domains. The LOV photocycle involves reversible formation of a photoproduct (LOV390) where the flavin mononucleotide (FMN) chromophore is covalently bound to a cysteine. LOV(390)thermally returns to the dark adapted state (LOV447) with a lifetime tau(rec)(s-to-h). In the LOV domain ofBacillus subtilis BsYtvA, the conserved F46 is one of the few residues undergoing a pronounced light-driven conformational change. PA and spectroscopic data show that in the YtvA variants F46A and F46Y light-induced structural changes are much smaller than those in the wild type (wt) protein,tau(rec)is strongly accelerated and the energy content of LOV(390)is lower for F46Y. MD simulations for each variant in the LOV(447)and LOV(390)states revealed an overall very stable structure of theBsYtvA-LOV domain. The largest variations emerged for the conserved HB network that includes FMN, Q123 (the flipping glutamine of LOV domains), and the conserved N104 and N94, with strong dependence on the presence of water. The lateral chain of Q123 in wt-LOV(447)can adopt three alternative conformations, and movements act in concert with F46 flexibility. In LOV390, Q123 remains instead fixed in the orientation adopted in the crystal structure. Interestingly, in F46A, Q123 is locked in a LOV447-like conformation (pseudo-dark-adapted state), in both LOV(447)and LOV390. In LOV(447)of F46Y the tyrosine hydroxyl group fixes a water molecule, which induces a Q123 conformation similar to wt-LOV390,i.e.a pseudo-photoproduct state. These pseudo-dark-adapted and photoproduct-like conformations of the Q123 sidechain may account for the strong acceleration of the photocycle in the two variants. Given the importance of the flipping glutamine in light-to-signal propagation in LOV proteins, the results presented here underscore a crucial structural and functional role of the conserved F46. MD results also indicate that F46 is not directly engaged in permeability of the FMN pocket, but is involved in solvent ordering and the formation of water bridges.