Aureochromes have recently been shown to act as blue-light-regulated transcription factors in the stramenopile alga Vaucheria frigida. They comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module with flavin mononucleotide (FMN) as a chromophore and a basic region leucine zipper (bZIP) domain as an effector. Aureochromes are the only members of a large LOV protein family, where the effector domain is located N-terminal to the sensor domain. This domain inversion positions the linking Jα helix of other LOV proteins to the terminus, raising the question of the role of Jα in aureochrome signaling. In phototropins, signaling proceeds from LOV2 via dissociation and unwinding of the Jα helix to the C-terminal kinase effector domain. In contrast, other LOV proteins have been demonstrated to activate the effector without the unfolding of Jα. We investigated the LOV domain of aureochrome1a from the diatom Phaeodactylum tricornutum both with and without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence that the Jα helix unfolds upon illumination. This unfolding is prerequisite for light-induced dimerization of LOV. Under illumination, full conversion to the dimer was observed by size exclusion chromatography. In the absence of the helix, a monomer was detected in the dark and in the light. As a further effect, the recovery of the dark state is 6-fold slower in LOV-Jα than LOV. We therefore postulate that the Jα helix plays an important role in aureochrome signaling.

Background: Plant cryptochromes are blue light sensors forming an exceptionally stable flavin neutral radical as a signaling state. Results: Blockage of proton transfer to flavin severely reduces the lifetime of the radical. Conclusion: The proton donor aspartic acid acts as an intrinsic stabilizer of the signaling state. Significance: The role of a key structural element is identified, distinguishing plant cryptochromes from other members of the family.

The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes’ light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical ‘aureo box’, TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity.

Flavoprotein radicals are important intermediates in many biochemical processes. In the blue light sensor plant cryptochrome, the radical state acts as a signaling state. An isolation and assignment of infrared bands of flavin radicals in the most relevant spectral region of carbonyl stretches is missing because of their overlap with absorption of water and the protein moiety. In this study, the neutral radical state of flavoproteins was investigated by Fourier transform infrared difference spectroscopy. The light-induced conversion of oxidized to neutral radical state was monitored in a plant cryptochrome and that of radical to fully reduced state in a DASH cryptochrome. A pure difference spectrum of flavin radical minus oxidized state was obtained from a point mutant of a phototropin LOV (light-, oxygen-, or voltage-sensitive) domain. The analysis of the spectra revealed a correlation between the frequencies of carbonyl vibrations of the flavin radical state and those of its visible absorption. Plant cryptochrome shows a very low frequency of the carbonyl stretch in the radical state. It is postulated that the downshift is caused by the charge of an adjacent aspartate, which donated its proton to flavin N(5). Contributions from the protein moiety to the spectra were isolated for DASH and plant cryptochromes. As a conclusion, the photosensitive domain of plant cryptochromes shows changes in secondary structure upon illumination, which might be related to signaling.

Aureochromes have been shown to act as blue-light-regulated transcription factors in algae in the absence of phototropins. Aureochromes comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module binding the flavin chromophore and a basic region leucine zipper (bZIP) domain as an effector. The domain arrangement in aureochromes with an N-terminal effector is inversed to other LOV proteins. To clarify the role of the linking A'α helix in signaling, we have investigated the LOV domain of aureochrome1a from the diatom alga Phaeodactylum tricornutum without the N-terminal A'α helix but with the C-terminal Jα helix. Results were analyzed in comparison to those previously obtained on the LOV domain with both flanking helices and on the LOV domain with the A'α helix but without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence by a band at 1656 cm(-1) that the A'α helix unfolds in response to light. This unfolding takes place only in the presence and as a consequence of the unfolding of the Jα helix, which points to an allosteric regulation. Size exclusion chromatography shows the LOV domain to be dimeric in the absence and monomeric in the presence of the A'α helix, implying that the folded helix covers the dimerization site. Therefore, the A'α helix directly modulates the oligomerization state of the LOV domain, whereas the Jα helix acts as an allosteric regulator. Both the allosteric control and the light-induced dimerization have not been observed in phototropin-LOV2 and point to a different signaling mechanism within the full-length proteins.