Structural And Functional Study Of Cyanobacterial Core-membrane Linker And A Phytochrome-like Protein

Light is a major ambient signal that regulates a wide range of important physiological processes in plants and cyanobacteria.Light can be used in several ways-it can it has an energy quality（e.g.,photosynthesis）or it can provide information（e.g.,biological photoreceptors）.Phycobiliproteins in the light-harvesting antennas（phycobilisomes）are the most abundant light-activated proteins in cyanobacteria.Phytochromes and their related proteins,the other group of bilin-based photoreceptors,were originally characterized as red/far-red photosensory receptors in green plants,but they have also been found in fungi,bacteria and cyanobacteria with a much wider range of absorbances.Phycobiliproteins and phytochromes are functionally and phylogenetically only distantly related;however,they not only possess very similar chromophores,but also share the same mode of covalent binding to and similar interactions with their apoproteins.Phycobilisomes,the light-harvesting antennas in cyanobacteria and red algae,consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker,and disks of phycocyanin and often also of phycoerythrin or phycoerythrocyanin that are arranged in long,externally extending rods.The core-membrane linker protein(L_CM),also called Apc E,is composed of an N-terminally located chromophore-binding domain and several C-terminal linker repeats that organize the phycobilisome core.This structure makes L_CM virtually insoluble and has prevented any detailed structural and functional analysis.Therefore,so far only the separate linker repeats have been crystallized,whereas for the chromophore domain of L_CM no structural data were available.Here,we report the crystal structure of a soluble chromophore domain construct of L_CM（Apc EΔ）from Nostoc sp.PCC7120 at 2.2?resolution.While its overall structure is similar to those of other phycobiliproteins,the covalently bound phycocyanobilin chromophore adopts a ZZZssa geometry,which differs from the ZZZasa geometry generally identified in other phycobiliproteins,but is identical to the geometry of phytochrome chromophores.The distance between two phycocyanobilins in a homodimer of the chromophore domain is 6.0 nm and that between two phycocyanobilins of adjacent homodimers is 3.2 nm,making the possibility of exciton coupling practically impossible.This assumption is supported by femto-second transient absorption spectroscopy.The light-harvesting proteins found in the phycobilisomes show excellent energy transfer among the chromophores that principally renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and extending into the near infrared.However,up to now,their application is limited,due to requirement simultaneous chromophore generation and by solubility problems.Based on our studies on the water-soluble chromophore-binding domain of L_CM,we fused this red-absorbing and fluorescing protein with a minimal chromophore-binding unit of phycocyanin.After double chromophorylation of both chromophore domains with phycocyanobilin（PCB）in E.coli,the fused phycobiliprotein domains absorbed light in the range of 610–660 nm,and fluoresced at～670 nm,similar to phycobilisomes devoid of phycoerythr（ocyan）in.The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin（PEB）,resulting in a chromoprotein absorbing around 540–575 nm,and fluorescing at～585 nm.The broad absorption bands and the large Stokes shifts render these chromoproteins candidates for imaging;they may also be helpful in studying FRET between phycobiliproteins and phycobilisome assembly.Recent discovery of cyanobacteriochromes（CBCR）further expands the family of bilin-based photoreceptors.These bilin-based signaling photoreceptors mediate physiological adaptations in response to light.As light-gated protein switches,signaling photoreceptors provide the basis for optogenetics.To understand in greater detail the photoconversion and signaling mechanism,we pursued parallel studies on two related phytochromes:Phy A from Arabidopsis thaliana（At Phy A）and Aph B,a phytochrome-like protein from Nostoc sp.PCC7120.The preliminary study on the three dimensional structure of Atphy A indicates the direction for further physiology study on signal transduction.The near-infared absorption and fluorescence property of Aph B offer significant advantages over currently popular fluorescence probes such as GFP for deep imaging in vivo and make it a primary target for light-controlled actuator modules in protein engineering of genetically encoded optogenetic tools.The ligand-binding GAF domains in cyanobacterial and plant phytochromes generally incorporate bilins as phytochrome chromophore.The genome of the cyanobacterium Nostoc sp.PCC7120 carries three genes（all4978,all7016 and alr7522）,encoding GAF proteins that were assumed,in contrast,to bind heme;functionally they were annotated as transcriptional regulators.They are composed of an N-terminal cofactor domain and a C-terminal helix-turn-helix（Ht H）motif.All4978 showed the highest affinity for protoheme binding.The heme binding capability of All7016 was moderate,and Alr7522 did not bind heme at all.The‘as isolated’form of All4978,identified as heme-containing by its Soret band（absorption maximum at 427 nm）,was assigned by electronic absorption,EPR-,and resonance Raman spectroscopy as a hexa-coordinated low spin-Fe^III heme with a distal cysteine ligand.The protoheme cofactor is non-covalently incorporated.Reduction of the heme could be accomplished by sodium dithionite,and also electro-spectrochemically;this latter method yielded remarkably low midpoint potentials of-445 and-453 m V（following Soret-andα-band absorption changes,respectively）.The reduced form of the heme(Fe^II state)binds NO and also CO.The cysteine coordination of the‘as isolated’Fe^III protein is evident,but interestingly,the reduced heme instead displays features indicative of histidine coordination.Cys-His ligand switches have been reported as a putative signaling mechanism in other heme-binding proteins,however,these novel cyanobacterial proteins are the first where such a ligand switch mechanism has been observed in a GAF domain.DNA-binding of the Ht H domain was investigated using a DNA sequence motif from its own promoter region.Formation of a protein-DNA complex could be demonstrated preferentially for the ferric state of the protein.