Introduction
Photosensory proteins give many organisms insights into their environment—providing windows unto their worlds, in a manner of speaking. Analogously, the ways that these proteins use small organic chromophores to convert photons into biological effects have provided windows through which biophysicists and biochemists have examined fundamental issues of protein-ligand interactions and allostery. The last ten years have seen significant advances in this field as new classes of photoreceptors have been identified and characterized. This continues in the May 18 issue of Science, in which Zoltowski et al.1 describe a powerful combination of biophysical and biochemical studies on Vivid, a member of the LOV (light-oxygen-voltage) domain class of blue light photoreceptors. This study provides critical insights into the mechanism Vivid uses to convert a light-induced configurational change at an internal FAD chromophore into a large conformational change on the protein surface.
LOV domains, a subset of the larger PAS (Per-ARNT-Sim) domain family2, serve as photosensors in bacteria, plants and fungi. Photosensing is dependent on light-sensitive flavin cofactors (FMN or FAD) that bind noncovalently within the cores of LOV domains. Upon illumination with blue light, a covalent bond is formed between a conserved cysteine residue and the C(4)a position on the flavin isoalloxazine ring. This covalent adduct exists for a finite period of time (seconds to hours, depending on the protein) before spontaneously decaying to the dark, noncovalent state. Since their initial characterization, LOV domains have been found in proteins with a wide variety of effector domains, ranging from histidine kinases to DNA binding motifs3.
The critical issue of how these effector domains are regulated by light activation has been best characterized so far with studies of plant phototropins, a class of blue-light-activated serine/threonine kinases. Early studies on phototropin LOV domains demonstrated that formation of the cysteine-C(4)a bond is accompanied by a significant conformational change in the surrounding protein4, 5, 6. This structural change has been described as a radical unfolding of a C-terminal 
-helix (named the J-helix) located outside the canonical mixed /
PAS domain core in Avena sativa phototropin1 LOV2 (ref. 5) (Fig. 1a). Replacing the critical cysteine residue within this LOV domain blocks the ability to generate the protein-flavin bond, displace the J-helix in vitro4, 5, and activate phototropin kinase activity in vivo7. Though details of the coupling between J-helix displacement and kinase activation remain under investigation, these findings and others in the field8, 9 clearly link LOV conformational changes with physiological responses to blue light.
Figure 1: LOV domain signaling.
(a) In phototropin. (b) In VVD. In both cases, a flavin chromophore is noncovalently bound within the mixed / PAS domain core in the dark (yellow), and noncanonical structural elements (blue) dock across the PAS -sheet. Illumination leads to the formation of a covalent adduct between a conserved cysteine residue and the flavin C(4)a position. This leads to a conformational change within the PAS core, which displaces or reorganizes the interactions with the noncanonical elements and triggers regulatory events with downstream effectors. WC-1 is white collar 1, a component of the heterodimeric WCC transcription factor that resets the Neurospora circadian clock in response to blue light.
Zoltowski et al. extend this foundation with their report on the fungal photoreceptor Vivid (VVD). This protein, which is predicted to contain only a LOV domain and small flanking sequences, helps the mold Neurospora crassa adapt to changes in environmental light levels10. This work provides the first structural data for VVD, including crystal structures of a truncated form (VVD-36) in the dark and lit states. These structures show that VVD adopts the expected mixed / fold surrounding an internal FAD chromophore and upon illumination forms the anticipated covalent adduct between the critical cysteine residue (Cys108) and the C(4)a position of the flavin cofactor. Interestingly, these structures also reveal that sequences outside of the canonical PAS domain core form a novel helix-strand cap (a-helix, b-strand) that packs against the central -sheet of the PAS core. Comparison of dark- and lit-state structures, acquired at 2.0 Å and 1.7 Å resolution, respectively, shows that illumination leads to conformational changes in the cap and PAS core, causing a "repacking" on the surface of the protein (Fig. 1b). Complementary gel filtration and small-angle X-ray scattering experiments suggest that the protein significantly expands when illuminated in solution, which is consistent with the observation from structural data that light triggers the displacement of residues N-terminal to the a -helix off the surface of the LOV domain.
To further test the linkage between cysteine-FAD bond formation and protein conformational changes, Zoltowski et al.1 used a combination of in vitro structural and in vivo functional studies. A focal point of this work is Cys71, which is located at the hinge region between the b-strand and the PAS core (Fig. 1b). Cys71 forms critical hydrogen bonds that bridge the a/b cap and the PAS core in the dark, but that are weakened in the light. The authors exploited this by generating point mutants at this position (for example, C71S) that are unable to convert into the expanded conformation in vitro after light-induced covalent adduct formation. Critically, in vivo complementation studies in N. crassa demonstrate that the C71S point mutation abolishes VVD's ability to regulate light-dependent responses (for example, carotenoid production), thereby establishing an important link between the conformational and functional changes triggered by photoactivation.
Two aspects of this work merit particular highlighting. First, these results establish strong parallels in the light-dependent modulation of interactions between the PAS core and cap structures in phototropin and VVD LOV domains. Despite significant differences in the cap structures and effectors, both systems clearly show that light activation triggers conformational changes in vitro that result in functional changes in vivo1, 5, 7. Second, mechanistic studies such as these benefit from a combination of experimental approaches. This strategy allowed Zoltowski et al.1 to identify light-induced motions in solution at a larger scale than those detected by X-ray crystallography, and it further allowed them to show the biological relevance of these motions.
Turning to the future, we are closer to answering critical questions about the mechanism used by LOV domains to regulate effector function. For phototropins, how J-helix unfolding is linked with kinase activation remains unclear; for VVD, light presumably alters its interaction with a yet-unidentified partner in the cell. More generally, LOV domains regulate a wide range of naturally occurring effectors3, and it remains to be determined whether the lessons learned from phototropins and VVD are generally applicable. In addition to providing insights into a diverse group of light-regulated proteins, better characterization of LOV domains may foster the development of new tools for biochemical and cell biological studies. The potential for this approach has already been demonstrated by the utility of several natural photosensors to spatially and temporally control changes in ion flux (for example, channel rhodopsin and NpHR; ref. 11) or cyclic AMP levels (for example, PAC; ref. 12) in several heterologous settings. Similar use of LOV-containing photosensors would allow the proteins that provide bacteria, fungi and plants with views of the world to open windows into entirely new research areas.
