Abstract
Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A′α helix is observed. With FMN, a “flip” of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead “swings” away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.
Highlights
Molecular dynamics (MD) simulations offer some support for the N5 protonation/ glutamine flip hypothesis,[33] and site-directed mutagenesis of the glutamine residue confirmed its vital importance for the function of distantly related LOV domains,[28,32] suggesting a common underlying mechanism
MD simulations of phototropin LOV domains generated a different conformation for the conserved glutamine side chain, altering the hydrogen bonding network to flanking helices.[35−37] Other recent reports propose that further glutamine side chain orientations are involved in LOV domain activation through hydrogen bonds with O4 of the flavin ring.[37−40] Given the importance of the potential hydrogen bonding associated with N5 of the flavin and the challenges associated with studying the lit state of thermally reverting LOV domains, we used 5-deazaflavin mononucleotide [5dFMN (Figure 1B)], an analogue that had previously been suggested to form a stable photochemical cysteinyl-flavin adduct in BsYtvA41 and successfully employed to alter the redox potentials of other flavoproteins.[42−44] At present, there are no experimental data to indicate whether the lit states of
OdAu1aLOV containing FMN reverted from its lit state to its dark state, with a half-life of 112 min, but no reversion was observed for OdAu1aLOV containing 5dFMN even after 7 days (Figure S1C,D)
Summary
Light-oxygen-voltage (LOV) photoreceptors are members of the Per-ARNT-Sim (PAS) superfamily of proteins that act as blue-light-sensing modules, mediating a wide range of processes, including phototropism, circadian rhythms, and stress responses.[1−7] The modular arrangement of sensory LOV domain proteins and effectors found in nature[4,5,8−10] has inspired many synthetic designs.[11−16] Such engineered proteins exhibit varying levels of photoresponsiveness, which can be partly attributed to the incomplete understanding of the mechanisms of allosteric control employed by natural LOV domains over effector modules.[12,16−19] To fully exploit the photochemical potential of LOV domains for engineered systems, a comprehensive picture of the structural determinants of allostery is needed. MD simulations of phototropin LOV domains generated a different conformation for the conserved glutamine side chain, altering the hydrogen bonding network to flanking helices.[35−37] Other recent reports propose that further glutamine side chain orientations are involved in LOV domain activation through hydrogen bonds with O4 of the flavin ring.[37−40] Given the importance of the potential hydrogen bonding associated with N5 of the flavin and the challenges associated with studying the lit state of thermally reverting LOV domains, we used 5-deazaflavin mononucleotide [5dFMN (Figure 1B)], an analogue that had previously been suggested to form a stable photochemical cysteinyl-flavin adduct in BsYtvA41 and successfully employed to alter the redox potentials of other flavoproteins.[42−44] At present, there are no experimental data to indicate whether the lit states of. We decided to examine the effect of 5dFMN incorporation on the photochemistry and function of Aureochrome1a (Au1a) of Ochromonas danica
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