Structural basis for gene regulation by a B12-dependent photoreceptor.

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter -35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

Light-dependent gene regulation by a coenzyme B12-based photoreceptor.

Cobalamin (B(12)) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B(12)-directed gene regulatory mechanisms via protein factors have, however, remained elusive. Recently, we reported down-regulation of a light-inducible promoter in the bacterium Myxococcus xanthus by two paralogous transcriptional repressors, of which one, CarH, but not the other, CarA, absolutely requires B(12) for activity even though both have a canonical B(12)-binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B(12) (5'-deoxyadenosylcobalamin, AdoB(12)), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB(12)-binding to the autonomous domain containing the B(12)-binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB(12) absorbs, dismantles active repressor oligomers by photolysing the bound AdoB(12) and weakens repressor-operator binding to allow transcription. By contrast, AdoB(12) alters neither CarA oligomerization nor operator binding, thus accounting for its B(12)-independent activity. Our findings unveil a functional facet of AdoB(12) whereby it serves as the chromophore of a unique photoreceptor protein class acting in light-dependent gene regulation. The prevalence of similar proteins of unknown function in microbial genomes suggests that this distinct B(12)-based molecular mechanism for photoregulation may be widespread in bacteria.

Analytical ultracentrifugation studies of oligomerization and DNA-binding of TtCarH, a Thermus thermophilus coenzyme B12-based photosensory regulator.

Thermus thermophilus transcriptional factor TtCarH belongs to a newly discovered class of photoreceptors that use 5'-deoxyadenosylcobalamin (AdoB12) as the light-sensing chromophore. Photoregulation relies on the repressor activity of AdoB12-bound oligomers in the dark, which light counteracts by oligomer disruption due to AdoB12 photolysis. In this study, we investigated TtCarH self-association and binding to DNA in the dark and in the light using analytical ultracentrifugation (AUC) methods, both sedimentation velocity (SV) as well as equilibrium (SE). From a methodological point of view, this study shows that AUC can provide hydrodynamic insights in cases where light is a crucial determinant of solution properties. For the light-sensitive TtCarH, absorbance as well as interference AUC data yielded comparable results. Sedimentation coefficients and whole-body hydrodynamic analysis from SV experiments indicate that in solution apo-TtCarH and light-exposed AdoB12-TtCarH are predominantly aspherical, ellipsoidal monomers, in accord with SE data. By comparison, AdoB12-TtCarH exists as a more compact tetramer in the dark, with smaller forms such as dimers or monomers remaining undetected and low levels of larger oligomers appearing at higher protein concentrations. AUC analyses indicate that in the dark AdoB12-TtCarH associates as a tetramer with DNA but forms smaller complexes in the apo form or if exposed to light. The self-association and DNA-binding properties of TtCarH deduced from AUC are consistent with data from size-exclusion and DNA-binding gel-shift assays. AUC analyses together with hydrodynamic modeling provide insights into the AdoB12- and light-dependent self-association and DNA-binding of TtCarH.

The photochemical mechanism of a B12-dependent photoreceptor protein.

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.