Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain Construct of the Cyanobacteriochrome Tlr0924 (SesA) without Stable Dimerization

Zeng

et al. 2020

Preprint

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Author contributionsXY designed the project; SB, SSM and XZ carried out cloning, purification, and crystallization; SB, XZ, SSM and NR conducted mutagenesis and spectroscopic experiments; SB, XZ, ZR, HS and XY collected monochromatic diffraction data; HS, CW and ZR collected Laue diffraction data; ZR processed the Laue data and carried out singlecrystal spectroscopic experiments; SB and XY determined, refined and analyzed crystal structures; NR carried out phylogenetic analysis; SB, XY, NR, MVM, and JCL interpreted the data; XY, NR, and JCL wrote the paper with inputs from all authors. Abstract (156 words)Cyanobacteriochromes are small, panchromatic photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. The molecular basis of far-red (FR) light perception by cyanobacteriochromes is currently unknown. Here we report the crystal structure of a far-red-sensing cyanobacteriochrome from Anabaena cylindrica PCC 7122, which exhibits a reversible far-red/orange photocycle.The 2.7 Å structure of its FR-absorbing dark state, determined by room temperature serial crystallography and cryo-crystallography, reveals an all-Z,syn configuration of its bound linear tetrapyrrole (bilin) chromophore that is less extended than the bilin chromophores of all known phytochromes. Based on structural comparisons with other bilin-binding proteins and extensive spectral analyses on mutants, we identify key proteinchromophore interactions that enable far-red sensing in bilin-binding proteins. We propose that FR-CBCRs employ two distinct tuning mechanisms, which work together to produce a large batho-chromatic shift. Findings of this work have important implications for development and improvement of photoproteins with far-red absorption and fluorescence. Significance Statement (99 words)Phytochromes are well known far-red-light sensors found in plants that trigger adaptive responses to facilitate competition for light capture with neighboring plants. Red-and farred-sensing are critical to cyanobacteria living in the far-red-enriched shade of plants.Here we report the crystal structure of a far-red-sensing cyanobacteriochrome, a distant cyanobacterial relative of phytochrome. These studies shed insight into the poorly understood molecular basis of far-red-sensing by phytobilin-based photoreceptors.Owing to the deep tissue penetration of far-red light, far-red-sensing photoreceptors offer promising protein scaffolds for developing gene-based photoswitches, optoacoustic contrast agents and fluorescent probes for in situ imaging and optogenetic applications.

Section: /Bodymentioning

confidence: 99%

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Bandara

Zeng

et al. 2020

Preprint

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“…Unlike canonical phytochromes, CBCR photosensory modules consist of one or more GAF domains that are sufficient for covalent attachment of bilin and photoconversion. These small CBCR domains have also been used as light-sensing modules in a variety of synthetic biology applications (27)(28)(29)(30)(31)(32). In contrast to canonical red/far-red phytochromes, CBCRs are able to sense light from near UV to far-red, utilizing a common phycocyanobilin (PCB) chromophore precursor (22)(23)(24)26).…”

mentioning

confidence: 99%

Crystal structure of a far-red–sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism

Bandara

Proc. Natl. Acad. Sci. U.S.A.

Zeng

et al. 2021

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Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red–sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red–sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red–absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein–chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

“…Purified protein (10 mM final concentration) in the 15E or 15Z states was added to initiate reaction in the dark. Photostates of protein were achieved using LEE filters (number 071 Tokyo blue and number 090 dark yellow green) as described previously (83), and confirmed via spectroscopic analysis. Assays were terminated after 1 h in the dark with addition of EDTA, pH 8.0, to a final concentration of 0.1 M. An aliquot (15 l) of each reaction solution was injected into an Agilent 1100 series HPLC and passed through a Phenomenex Synergi 4 M Fusion-RP 80 Å column and Security Guard Cartridge (C18, 4 ϫ 2 mm), under conditions previously described (83).…”

Section: In Vitro Cyclase Assaymentioning

confidence: 99%

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells

Blain-Hartung

Journal of Biological Chemistry

Moreno

et al. 2018

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Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium sp. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.