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Phytochromes (PHYs) are photoreceptor proteins first discovered in plants, where they control a variety of photomorphogenesis events. PHYs as photochromic proteins can reversibly switch between two distinct states: a red light (Pr) and a far-red light (Pfr) absorbing form. The discovery of Bacteriophytochromes (BphPs) in nonphotosynthetic bacteria has opened new frontiers in our understanding of the mechanisms by which these natural photoswitches can control single cell development, although the role of BphPs in vivo remains largely unknown. BphPs are dimeric proteins that consist of a photosensory core module (PCM) and an enzymatic domain, often a histidine kinase. The PCM is composed of three domains (PAS, GAF, and PHY). It holds a covalently bound open-chain tetrapyrrole (biliverdin, BV) chromophore. Upon absorption of light, the double bond between BV rings C and D isomerizes and reversibly switches the protein between Pr and Pfr states. We report crystal structures of the wild-type and mutant (His275Thr) forms of the canonical BphP from the nonphotosynthetic myxobacterium Stigmatella aurantiaca (SaBphP2) in the Pr state. Structures were determined at 1.65 Å and 2.2 Å (respectively), the highest resolution of any PCM construct to date. We also report the room temperature wild-type structure of the same protein determined at 2.1 Å at the SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Our results not only highlight and confirm important amino acids near the chromophore that play a role in Pr-Pfr photoconversion but also describe the signal transduction into the PHY domain which moves across tens of angstroms after the light stimulus.
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Structural basis for light control of cell development revealed by crystal structures of a myxobacterial phytochrome
Phytochromes are red-light photoreceptors that were first characterized in plants, with homologs in photosynthetic and non-photosynthetic bacteria known as bacteriophytochromes (BphPs). Upon absorption of light, BphPs interconvert between two states denoted Pr and Pfr with distinct absorption spectra in the red and far-red. They have recently been engineered as enzymatic photoswitches for fluorescent-marker applications in non-invasive tissue imaging of mammals. This article presents cryo- and room-temperature crystal structures of the unusual phytochrome from the non-photosynthetic myxobacteriumStigmatella aurantiaca(SaBphP1) and reveals its role in the fruiting-body formation of this photomorphogenic bacterium. SaBphP1 lacks a conserved histidine (His) in the chromophore-binding domain that stabilizes the Pr state in the classical BphPs. Instead it contains a threonine (Thr), a feature that is restricted to several myxobacterial phytochromes and is not evolutionarily understood. SaBphP1 structures of the chromophore binding domain (CBD) and the complete photosensory core module (PCM) in wild-type and Thr-to-His mutant forms reveal details of the molecular mechanism of the Pr/Pfr transition associated with the physiological response of this myxobacterium to red light. Specifically, key structural differences in the CBD and PCM between the wild-type and the Thr-to-His mutant involve essential chromophore contacts with proximal amino acids, and point to how the photosignal is transduced through the rest of the protein, impacting the essential enzymatic activity in the photomorphogenic response of this myxobacterium.
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- PAR ID:
- 10588838
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- IUCrJ
- Date Published:
- Journal Name:
- IUCrJ
- Volume:
- 5
- Issue:
- 5
- ISSN:
- 2052-2525
- Page Range / eLocation ID:
- 619 to 634
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light–absorbing Pr state and a far-red light–absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo–electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.more » « less
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Phytochromes are red-light photoreceptors first identified in plants, with homologs found in bacteria and fungi, that regulate a variety of critical physiological processes. They undergo a reversible photocycle between two distinct states: a red-light-absorbing Pr form and a far-red light-absorbing Pfr form. This Pr/Pfr photoconversion controls the activity of a C-terminal enzymatic domain, typically a histidine kinase (HK). However, the molecular mechanisms underlying light-induced regulation of HK activity in bacteria remain poorly understood, as only a few structures of unmodified bacterial phytochromes with HK activity are known. Recently, cryo-EM structures of a wild-type bacterial phytochrome with HK activity are solved that reveal homodimers in both the Pr and Pfr states, as well as a heterodimer with individual monomers in distinct Pr and Pfr states. Cryo-EM structures of a truncated version of the same phytochrome—lacking the HK domain—also show a homodimer in the Pfr state and a Pr/Pfr heterodimer. Here, we describe in detail how structural information is obtained from cryo-EM data on a full-length intact bacteriophytochrome, and how the cryo-EM structure can contribute to the understanding of the function of the phytochrome. In addition, we compare the cryo-EM structure to an unusual x-ray structure that is obtained from a fragmented full-length phytochrome crystallized in the Pr-state.more » « less
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Abstract Myxobacteria are non-photosynthetic bacteria distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies that are formed in response to nutrient deprivation and stimulated by light. Here, we report an entrained, rhythmic pattern ofMyxococcus macrosporusfruiting bodies, forming consistently spaced concentric rings when grown in the dark. Light exposure disrupts this rhythmic phenotype, resulting in a sporadic arrangement and reduced fruiting-body count.M. macrosporusgenome encodes a red-light photoreceptor, a bacteriophytochrome (BphP), previously shown to affect the fruiting-body formation in the related myxobacteriumStigmatella aurantiaca. Similarly, the formation ofM. macrosporusfruiting bodies is also impacted by the exposure to BphP—specific wavelengths of light. RNA-Seq analysis ofM. macrosporusrevealed constitutive expression of thebphPgene. Phytochromes, as light-regulated enzymes, control many aspects of plant development including photomorphogenesis. They are intrinsically correlated to circadian clock proteins, impacting the overall light-mediated entrainment of the circadian clock. However, this functional relationship remains unexplored in non-photosynthetic prokaryotes. Genomic analysis unveiled the presence of multiple homologs of cyanobacterial core oscillatory gene,kaiC, in various myxobacteria, includingM. macrosporus,S. aurantiaca and M. xanthus. RNA-Seq analysis verified the expression of allkaiChomologs inM. macrosporusand the closely relatedM. xanthus, which lacksbphPgenes. Overall, this study unravels the rhythmic growth pattern duringM. macrosporusdevelopment, governed by environmental factors such as light and nutrients. In addition, myxobacteria may have a time-measuring mechanism resembling the cyanobacterial circadian clock that links the photoreceptor (BphP) function to the observed rhythmic behavior. Graphical abstractmore » « less
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Light signals perceived by a group of photoreceptors have profound effects on the physiology, growth, and development of plants. The red/far-red light–absorbing phytochromes (phys) modulate these aspects by intricately regulating gene expression at multiple levels. Here, we report the identification and functional characterization of an RNA-binding splicing factor, SWAP1 (SUPPRESSOR-OF-WHITE-APRICOT/SURP RNA-BINDING DOMAIN-CONTAINING PROTEIN1). Loss-of-function swap1-1 mutant is hyposensitive to red light and exhibits a day length–independent early flowering phenotype. SWAP1 physically interacts with two other splicing factors, (SFPS) SPLICING FACTOR FOR PHYTOCHROME SIGNALING and (RRC1) REDUCED RED LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1 in a light-independent manner and forms a ternary complex. In addition, SWAP1 physically interacts with photoactivated phyB and colocalizes with nuclear phyB photobodies. Phenotypic analyses show that the swap1sfps , swap1rrc1, and sfpsrrc1 double mutants display hypocotyl lengths similar to that of the respective single mutants under red light, suggesting that they function in the same genetic pathway. The swap1sfps double and swap1sfpsrrc1 triple mutants display pleiotropic phenotypes, including sterility at the adult stage. Deep RNA sequencing (RNA-seq) analyses show that SWAP1 regulates the gene expression and pre–messenger RNA (mRNA) alternative splicing of a large number of genes, including those involved in plant responses to light signaling. A comparative analysis of alternative splicing among single, double, and triple mutants showed that all three splicing factors coordinately regulate the alternative splicing of a subset of genes. Our study uncovered the function of a splicing factor that modulates light-regulated alternative splicing by interacting with photoactivated phyB and other splicing factors.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1107/s2052252518010631
