Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Abstract

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.

An automated platform for in situ serial crystallography at room temperature

“…The first system is a novel far-red photoreceptor that confers reversible photoconversion between the far-redabsorbing (Pfr) state and orange-absorbing (Po) state based on a single bilin-binding GAF domain (Bandara et al, 2020). Since our photoreceptor construct corresponds to the third GAF domain of a multi-domain sensory histidine kinase from Anabaena cylindrica PCC 7122, we refer to this crystal as G3 hereafter.…”

“…which are as photoactive as in solution (Bandara et al, 2020). Using conventional cryocrystallography, we obtain crystallography datasets of G3 in the space group P4 2 22 up to 2.9 Å resolution.…”

“…We carry out in situ serial diffraction experiments using 22 crystallization devices, from which we obtain a Laue dataset of nearly 50-fold redundancy. Structure refinement against this room-temperature Laue dataset gives rise to a well resolved electron-density map with no gaps in the protein backbone, which enables confident interpretation of a highly unusual and important structure (Table S1 of the supporting information) (Bandara et al, 2020).…”

“…G3 in the Pfr state is crystallized following pre-illumination with filtered green light at wavelengths of 550 AE 20 nm (Newport) for 15 min before setting up the crystallization (Bandara et al, 2020). The Pfr crystals grow in the dark at room temperature using the hanging-drop vapor-diffusion method by mixing the protein solution (3 mg ml À1 ) and crystallization solution (17% PEG 10 000) and 0.1 M ammonium acetate in the buffer of 0.1 M bis-Tris at pH 5.5 with yttrium research papers (III) chloride as an additive.…”

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

“…The red/green subfamily harbors a fully protonated chromophore in both photostates, but a twisted conformation of the A-and Drings is responsible for the blue-shifted photoproduct (8,(20)(21)(22)(23); this is known as the "trappedtwist photocycle" (24). Recently, Bandara et al reported the structure of a far-red absorbing state of a CBCR that adopts a atypical C15-Z,syn configuration (25). They proposed a model that its far-red absorption maximum arises from a cationic bilin tautomer in which the A-ring is formally di-protonated while the B-ring remains deprotonated.…”