Junrui Zhang

High resolution structure of deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution.

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXα. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45Å resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C32 carbon of biliverdin to Cys24, the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.

Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

Structural basis for the photoconversion of a phytochrome to the activated Pfr form

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB′. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C31 carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.

Quaternary organization of a phytochrome dimer as revealed by cryoelectron microscopy

Phytochromes are a collection of dimeric photoreceptors that direct a diverse array of responses in plants and microorganisms through photoconversion between a red light-absorbing ground state Pr, and a far-red light-absorbing photoactivated state Pfr. Photoconversion from Pr to Pfr is initiated by a light-driven rotation within the covalently attached bilin, which then triggers a series of protein conformational changes in the binding pocket. These movements ultimately affect an appended output module, which often has reversible protein kinase activity. Propagation of the light signal from the bilin to the output module likely depends on the dimerization interface but its architecture and response to phototransformation remain unclear. Here, we used single particle cryoelectron microscopy to determine the quaternary arrangement of the phytochrome dimer as Pr, using the bacteriophytochrome (BphP) from Deinococcus radiodurans. Contrary to the long-standing view that the two monomers are held together solely via their C-terminal region, we provide unambiguous evidence that the N-terminal bilin-binding region of BphP also provides a dimerization interface with the C-terminal kinase domain appearing as a more flexible appendage. The BphP monomers dimerize in parallel with the polypeptides intimately twisting around each other in a right-handed fashion. Based on this electron microscopic picture, we propose that the light-driven conformational changes transmitted from the chromophore to the output module along the spine of this extensive dimer interface is the central feature underpinning phytochrome signaling.

Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B′, that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. 1H-15N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

Phytochrome signaling: solving the Gordian knot with microbial relatives.

Phytochromes encompass a diverse collection of biliproteins that regulate numerous photoresponses in plants and microorganisms. Whereas the plant versions have proven experimentally intractable for structural studies, the microbial forms have recently provided important insights into how these photoreceptors work at the atomic level. Here, we review the current understanding of these microbial phytochromes, which shows that they have a modular dimeric architecture that propagates light-driven rotation of the bilin to distal contacts between adjacent signal output domains. Surprising features underpinning this signaling include: a deeply buried chromophore; a knot and hairpin loop that stabilizes the photosensing domain; and an extended helical spine that translates conformational changes in the photosensing domain to the output domain. Conservation within the superfamily both in modular construction and sequence strongly suggests that higher plant phytochromes work similarly as light-regulated toggle switches.

Structure-Guided Engineering of Plant Phytochrome B with Altered Photochemistry and Light Signaling

Modification of phytochrome photochemistry and behavior by structure-guided mutagenesis provides new tools to study signaling and manipulate photomorphogenesis for potential agricultural benefit. Phytochromes (phys) encompass a diverse collection of biliproteins that enable cellular light perception by photoconverting between a red-light-absorbing ground state (Pr) and a far-red light-absorbing active state (Pfr). Based on the central role of plant phys in controlling numerous agriculturally important processes, their rational redesign offers great promise toward accelerating crop improvement. Employing as templates the available three-dimensional models of the photosensory module within bacterial phys, we report here our initial attempt to apply structure-guided mutagenesis to phy engineering using Arabidopsis (Arabidopsis thaliana) phyB, the dominant isoform in light-grown plants, as the example. A collection of phyB mutants was generated affecting the bilin-binding pocket that altered photochemistry, thermal stability, and/or nuclear localization patterns, some of which also impacted phenotypic outputs. Of particular interest are the Y361F substitution, which created Arabidopsis plants with greatly enhanced light sensitivity, mutants variably altered in Pfr-to-Pr thermal reversion and nuclear aggregation, and the D307A substitution, which failed to photoconvert from Pr to Pfr and display light-induced nuclear aggregation but retained some biological activity and accelerated turnover in red light. Taken together, this collection provides variants potentially useful to agriculture as well as new tools to better understand the molecular mechanisms underpinning phy signaling.