Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level.
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ABSTRACT:
SUBMITTER: Otero LH
PROVIDER: S-EPMC8612531 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Light sensing mechanisms allow prokaryotes and eukaryotes to adapt to constantly changing environmental factors. Phytochromes constitute a widespread biological photoreceptor family that typically interconvert between two photostates called Pr (red light-absorbing) and Pfr (far-red light-absorbing). Despite the vast structural information reported on phytochromes, the lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly of the protein. These structural findings, combined with mutational, biochemical and computational studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level. As the transduction mechanism herein reported involves elements highly conserved in other phytochromes, it might be extended to other members of this family of photoreceptors.
2021-12-17 | PXD026829 | Pride
Project description:Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes.
| S-EPMC4982709 | biostudies-literature
Project description: Not available
| EMPIAR-12093 | biostudies-other
Project description: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.
| S-EPMC2807988 | biostudies-literature
Project description:We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1-595, including the NTE (N-terminal extension), PAS (Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) and PHY (phytochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with 13C- and 15N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D -ring in a ?-facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the ?-facial ring D . Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D -ring Za?Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B -ring propionate partner swap from R317 to R287, changes in the C -ring propionate hydrogen-bonding network, breakage of the D272-R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326-D272-S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A . We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.
| S-EPMC5928327 | biostudies-literature
Project description:Femtosecond photodynamics of the Pfr form of the red/far-red phytochrome N-terminal PAS-GAF-PHY photosensory core module of the cyanobacterial phytochrome Cph1 (termed Cph1Δ) from Synechocystis were resolved with visible broadband transient absorption spectroscopy. Multiphasic generation dynamics via global target analysis revealed parallel evolution of two pathways with distinct excited- and ground-state kinetics. These measurements resolved two subpopulations: a majority subpopulation with fast excited-state decay and slower ground-state dynamics, corresponding to previous descriptions of Pfr dynamics, and a minority subpopulation with slower excited-state decay and faster ground-state primary dynamics. Both excited-state subpopulations generated the isomerized, red-shifted Lumi-Ff photoproduct (715 nm); subsequent ground-state evolution to a blue-shifted Meta-Fr population (635 nm) proceeded on 3 ps and 1.5 ns time scales for the two subpopulations. Meta-Fr was spectrally similar to a recently described photoinactive fluorescent subpopulation of Pr ((Fluor)Pr). Thus, the reverse Pfr to Pr photoconversion of Cph1Δ involves minor structural deformation of Meta-Fr to generate the fluorescent, photochemically refractory form of Pr, with slower subsequent equilibration with the photoactive Pr subpopulation ((Photo)Pr).
| S-EPMC4184438 | biostudies-literature
Project description:The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Salmonella and Escherichia coli, the primary subspecies classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are, therefore, of interest for pathogen surveillance. Here, we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping Salmonella enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S rRNA gene sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intraspecies variation and direct sequencing from environmental samples could be valuable.IMPORTANCEIn order to prevent and stop outbreaks of foodborne pathogens, it is important that we can detect when pathogenic bacteria are present in a food or food-associated site and identify connections between specific pathogenic bacteria present in different samples. In this work, we develop a new computational technology that allows the important foodborne pathogens Escherichia coli and Salmonella enterica to be serotyped (a subspecies level classification) from sequencing of a single-marker gene, and the 16S rRNA gene often used to surveil bacterial communities. Our results suggest current limitations to serotyping from 16S rRNA gene sequencing alone but set the stage for further progress that we consider likely given the rapid advance in the long-read sequencing technologies and genomic databases our work leverages. If this research direction succeeds, it could enable better detection of foodborne pathogens before they reach the public and speed the resolution of foodborne pathogen outbreaks.
| S-EPMC10949465 | biostudies-literature
Project description:The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Escherichia coli and Salmonella, the primary sub-species classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are therefore of interest for pathogen surveillance. Here we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping S. enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data, and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intra-species variation and direct sequencing from environmental samples could be valuable.
| S-EPMC10327058 | biostudies-literature
Project description:Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a "flip-and-rotate" model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.
| S-EPMC2747172 | biostudies-literature
Project description:Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.
| S-EPMC7809406 | biostudies-literature