A far-red cyanobacteriochrome lineage specific for verdins Marcus V. Morenoa, Nathan C. Rockwella, Manuel Moraa, Andrew J. Fishera,b, and J. Clark Lagariasa,1 aDepartment of Molecular and Cellular Biology, University of California, Davis, CA 95616; and bDepartment of Chemistry, University of California, Davis, CA 95616 Contributed by J. Clark Lagarias, September 29, 2020 (sent for review July 30, 2020; reviewed by Wolfgang Gärtner and Matias D. Zurbriggen) Cyanobacteriochromes (CBCRs) are photoswitchable linear tetra- phytochrome interaction factors has been used to control gene pyrrole (bilin)-based light sensors in the phytochrome superfamily transcription, actin assembly, and GTP-dependent signaling with a broad spectral range from the near UV through the far red modules using light (14–16). Photoreversible transcriptional (330 to 760 nm). The recent discovery of far-red absorbing CBCRs control was also achieved in bacteria using the light input domain (frCBCRs) has garnered considerable interest from the optogenetic of cyanobacterial phytochrome 1 (Cph1) from Synechocystis sp. and imaging communities because of the deep penetrance of far- PCC 6803 fused to the receiver output domain of OmpR red light into mammalian tissue and the small size of the CBCR (17–19). Extensive engineering of bacterial phytochromes protein scaffold. The present studies were undertaken to deter- (BphPs) has yielded red-absorbing and far-red–emitting fluo- mine the structural basis for far-red absorption by JSC1_58120g3, a rescent proteins (20–24). BphPs provide a useful template for frCBCR from the thermophilic cyanobacterium Leptolyngbya sp. such applications because they incorporate more extensively JSC-1 that is a representative member of a phylogenetically dis- conjugated and a longer wavelength-absorbing biliverdin IXα tinct class. Unlike most CBCRs that bind phycocyanobilin (PCB), a (BV) chromophore instead of the reduced phycobilins phyco- phycobilin naturally occurring in cyanobacteria and only a few cyanobilin (PCB) and phytochromobilin (PΦB) used by cyano- eukaryotic phototrophs, JSC1_58120g3’s far-red absorption arises 1 2 bacterial, algal, and plant phytochromes (SI Appendix, Fig. S1A). from incorporation of the PCB biosynthetic intermediate 18 ,18 - Φ 1 2 PCB and P B are restricted to oxygenic photosynthetic taxa, dihydrobiliverdin (18 ,18 -DHBV) rather than the more reduced whereas BV is present in a wider range of organisms including and more abundant PCB. JSC1_58120g3 can also yield a far- – mammals (9, 25). BIOCHEMISTRY red absorbing adduct with the more widespread linear tetrapyr- Canonical phytochromes, which include BphPs and plant/ role biliverdin IXα (BV), thus circumventing the need to coproduce cyanobacterial phytochromes, are mostly dimeric proteins with a or supplement optogenetic cell lines with PCB. Using high- conserved, N-terminal PAS-GAF-PHY tridomain photosensory resolution X-ray crystal structures of 181,182-DHBV and BV adducts core module of ∼500 amino acids. Attempts to create smaller, of JSC1_58120g3 along with structure-guided mutagenesis, we have defined residues critical for its verdin-binding preference monomeric fluorescent probes based on BphPs have been suc- and far-red absorption. Far-red sensing and verdin incorporation cessful, but the most robust of these have substantially blue- make this frCBCR lineage an attractive template for developing shifted spectra (26). The discovery of cyanobacteriochromes robust optogenetic and imaging reagents for deep tissue (CBCRs), distantly related bilin-binding GAF-only photorecep- BIOPHYSICS AND COMPUTATIONAL BIOLOGY applications. tors that sense light ranging from the near UV to the far red (27–36), has proven an exciting opportunity for engineering biliprotein | photochromism | bilin-protein interaction | spectral tuning | smaller optogenetic reporters and control elements. Using phylogenic analysis Significance early all organisms utilize photosensory proteins to detect Nand respond to the light environment. In many animals, Cyanobacteriochrome (CBCR) photoreceptors leverage light- vision is mediated by type II opsin photoreceptors utilizing a triggered photoisomerization of bound bilin chromophores to retinal chromophore (1). Type I opsins include light-gated pro- regulate gene expression, directional movement, and cell–cell ton and ion channels in archaea, bacteria, green algae, and fungi communication in cyanobacteria. CBCRs responding to far-red (2, 3). Entrainment of circadian rhythms to light/dark cycles can light provide an emerging class of small reagents for applica- tions in synthetic biology. We have found CBCRs that use involve retinal-based opsins (4), flavin-based cryptochromes (5), 1 2 or light-oxygen-voltage-sensing (LOV) domains (6, 7). LOV 18 ,18 -dihydrobiliverdin or biliverdin chromophore precursors domains are also found in phototropins, the photoreceptors to sense far-red light. This lineage is distinguished from others controlling plant phototropism (8). Linear tetrapyrrole (bilin)- by exclusion of phycobilins, the normal CBCR chromophores. based phytochrome photoreceptors are widespread in plants, Owing to the deep penetrance of far-red light in mammalian algae, fungi, cyanobacteria, and other bacteria (9, 10). Plant tissue and availability of biliverdin therein, this lineage of phytochromes use 15,16 double-bond photoisomerization of CBCRs holds great potential for developing synthetic biology their bilin chromophores to photoconvert between a dark- tools of biomedical interest. adapted 15Z red-absorbing photostate (P )anda15E far-red– r Author contributions: M.V.M., N.C.R., A.J.F., and J.C.L. designed research; M.V.M., N.C.R., absorbing (Pfr) photoproduct, regulating diverse developmental M.M., and A.J.F. performed research; M.V.M., N.C.R., and M.M. contributed new reagents/ processes including seed germination, chloroplast develop- analytic tools; M.V.M., N.C.R., A.J.F., and J.C.L. analyzed data; and M.V.M., N.C.R., and ment, seedling establishment, shade avoidance, and flowering J.C.L. wrote the paper. (9, 11, 12). Reviewers: W.G., University of Leipzig; and M.D.Z., University of Düsseldorf. Members of the phytochrome superfamily are attractive tar- The authors declare no competing interest. gets for development of optogenetic and imaging tools due to Published under the PNAS license. their strong absorption in the near-infrared window that provides 1To whom correspondence may be addressed. Email: [email protected]. optimal light penetrance and minimal interference from cellular This article contains supporting information online at https://www.pnas.org/lookup/suppl/ components in mammalian tissue (13). Reversible red- and far- doi:10.1073/pnas.2016047117/-/DCSupplemental. red–mediated binding of Arabidopsis thaliana phytochrome B to www.pnas.org/cgi/doi/10.1073/pnas.2016047117 PNAS Latest Articles | 1of9 Downloaded by guest on September 28, 2021 structure-guided and random mutagenesis approaches, two JSC1_58120g3 as a query identified a number of CBCR se- groups have developed BV-binding CBCRs for fluorescent ap- quences with characteristic amino acid substitutions relative to plications in the far-red spectral region (37, 38). Although these canonical red/green CBCRs, with other residues conserved engineered proteins have lower fluorescence quantum yields among the two groups (SI Appendix, Fig. S2A). Phylogenetic than engineered BV-binding phycobiliproteins (39–42), fluores- analysis established that JSC1_58120g3 and related proteins cent CBCRs may yet hold promise as probes for superresolution comprise a clade that arose within the XRG lineage. These microscopy due to the combination of photo-switchable and – proteins are distinct from the AmBV4 family (Fig. 1A and SI long-lasting fluorescence (43). Current far-red sensing CBCRs Appendix, Fig. S2B). As members of the XRG lineage, (frCBCRs) perhaps hold greater promise as optogenetic actua- JSC1_58120g3 and other members of this lineage are also not tors and photoacoustic contrast agents that photoconvert under closely related to the previously identified PCB-binding FRoGGR far-red light to yield light-regulated responses. Indeed, CBCR- regulated histidine kinases (cHKs) (18, 19, 44) and CBCR- cluster that evolved within the greater green/red (GGR) lineage of regulated photoswitchable adenylyl cyclases (cPACs) (45) have CBCRs (52). been exploited to control gene expression in cells using green/ We next expressed two additional proteins within this lineage Φ red, UV/green, blue/green, and far-red/red photocycles. in Escherichia coli cells engineered for PCB or P B synthesis. Two phylogenetically distinct clusters of naturally occurring Both Mic7113_1903g4 and AFZ15460g4 yielded photoactive frCBCRs were identified recently. Members of the first cluster of CBCRs with FR/R photocycles similar to those of JSC1_58120g3 frCBCRs have been reported only from Acaryochloris marina (Fig. 1 B–G and SI Appendix, Table S1). Dark-state spectra for MBIC 11017. These frCBCRs detect far-red light due to their ability to incorporate BV in the absence of PCB (37, 46–49). This frCBCR clade arose within the eXtended Red/Green (XRG) JSC1_58120g3 A MC7420_3507g3 CBCR lineage, and representative members typically exhibit far- DPYLoar AFZ15460g4 no proline red/orange (FR/O) photocycles when bound to BV (15Z λmax ∼ AFZ15460g6 AmBV4 697 to 702 nm) and red/green photocycles when bound to PCB red/blue Mic7113_1903g3 (47–49). Based on four conserved residues sufficient to confer red/green Mic7113_1903g4