Light-dependent flavin redox and adduct states control the conformation and DNA binding activity of the transcription factor EL222
The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of -helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. Significance StatementFlavin-binding light-oxygen-voltage (LOV) proteins constitute a prominent example of highly evolved chromophore-containing proteins that convert light into biochemical changes in the cell. However, it is not well understood how blue-light orchestrates changes in LOV structure and function. Here we show that the dynamics, oligomerization and DNA-binding properties of the photocontrolled transcription factor EL222 are dependent on both the flavin redox state and thioadduct formation. In the dark, monomeric EL222 forms transient encounter complexes with DNA. Under illumination, two distinct lit states are sequentially generated, termed lit1 and lit2, that are both able to assemble into EL222:DNA (2:1) complexes. Our results reveal the coupling between flavin photochemistry (protonation and covalent linkage) and fold stability in EL222 and potentially other flavoproteins.