A Noncanonical Chromophore Reveals Structural Rearrangements of the Light-Oxygen-Voltage Domain upon Photoactivation

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.

Figure 1

(A) Formation of a cysteinyl-FMN covalent adduct occurs upon absorption of blue light by flavin mononucleotide (FMN). Spontaneous thermal reversion re-forms the dark-adapted state. (B) Structure of 5-deazaflavin mononucleotide (5dFMN) with a carbon atom (blue) at position 5. (C) Domain topology of O. danica Aureochrome1a. Au1a_bZIPLOV comprises bZIP and LOV domains, and Au1a_LOV comprises only the LOV domain. UV–vis spectra of thermal reversion from the lit to dark state of (D) FMN-containing (red–green) and (E) 5dFMN-containing (orange–blue) OdAu1a_LOV. Spectra were recorded every hour for the first 3 h and then every 2 h. Reversion kinetics were monitored at 448 nm for FMN-containing OdAu1a_LOV and 406 nm for 5dFMN-containing OdAu1a_LOV. Lit-state FMN OdAu1a_LOV reverts to its dark state with a half-life of 112 min. No reversion to the dark state is observed for lit-state 5dFMN-containing OdAu1a_LOV.

Figure 2

Circular dichroism spectra of (A) FMN-containing and (B) 5dFMN-containing OdAu1a_LOV (20 μM) in potassium phosphate buffer (10 mM, pH 7.0) under dark and light (450 nm) conditions. Green and red traces correspond to dark and lit states of FMN, respectively, while blue and orange traces correspond to dark and lit states of 5dFMN, respectively. Size-exclusion chromatography of OdAu1a_LOV. (C) FMN-containing OdAu1a_LOV under dark (green) and illuminated (red dashed) conditions. (D) 5dFMN-containing OdAu1a_LOV under dark (blue) and illuminated (orange dashed) conditions. Electrophoresis mobility shift assays of OdAu1a_bZIPLOV with a DNA target (40 nM) for (E) illuminated FMN-containing OdAu1a_bZIPLOV, (F) dark-state FMN-containing OdAu1a_bZIPLOV, (G) illuminated 5dFMN-containing OdAu1a_bZIPLOV, and (H) dark-state 5dFMN-containing OdAu1a_bZIPLOV. The first lane contains TAMRA-labeled DNA only, and subsequent lanes have increasing protein concentrations (from 0.4 to 12 μM from left to right, respectively).

Figure 3

Dimer arrangements for X-ray crystal structures of FMN- and 5dFMN-containing OdAu1a_LOV under dark (left), illuminated (middle), and light-grown (right) conditions. (A) The 1.37 Å structure of dark-state FMN-containing OdAu1a. The asymmetric unit contained four monomers as parallel dimers (green) with A′α positioned across the β-sheet surface (half black box). Loops of each monomer lie close to each other (black curved line). (B) The 1.50 Å structure of illuminated crystals of dark-grown FMN-containing OdAu1a. The asymmetric unit contained a single monomer (pink), forming a parallel dimer similar to that in the dark state when considering a symmetry equivalent (gray). (C) The 1.66 Å structure of light-grown FMN-containing OdAu1a featuring a unique dimer arrangement with A′α being repositioned across a β-sheet surface (black box) and loop region rearrangement (dashed arrow). (D) The 1.97 Å structure of dark-state 5dFMN-containing OdAu1a with a dimer similar to dark-state FMN. (E) The 1.43 Å structure of illuminated 5dFMN-containing OdAu1a with a symmetry partner equivalent to a dimer colored gray. (F) The 2.00 Å structure of light-grown 5dFMN-containing OdAu1A showing a similar loop (black curved line) and A′α helix arrangement (black box) as for dark-state and illuminated proteins with a symmetry equivalent colored gray.

Figure 4

Electron density maps (gray mesh) for FMN or 5dFMN, Cys230, and residues forming hydrogen bonding networks among O4 of FMN, Asn272, Gln293, and Asn194 are displayed at the σ = 1 level. Partial occupancies are colored by characteristic conformations observed for dark-state (green), illuminated (orange), or light-grown (purple) structures. Yellow dashed lines indicate predicted hydrogen bonding. (A) Dark-state FMN (1.36 Å). (B) Illuminated FMN (1.50 Å). (C) Light-grown FMN (1.67 Å). (D) Dark-state 5dFMN (1.97 Å). (E) Illuminated 5dFMN (1.43 Å). (F) Light-grown 5dFMN (2.00 Å).

Figure 5

In the dark-state conformation (green), conserved Gln293 hydrogen bonds to O4 and N5 of FMN. Illumination with blue light results in the Gln293 “swing” state (orange) where its side chain rotates away from the FMN chromophore. Progression to the Gln293 “flip” state (purple) may occur from the “swing” state or from the dark state but cannot proceed when the protein is trapped in the crystal lattice. Rotation of the side chain of Asn194 upon formation of the “flip” state is likely to lead to conformational changes in the A′α helix.