| 1. |
- Woitowich, N. C., et al.
(författare)
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Structural basis for light control of cell development revealed by crystal structures of a myxobacterial phytochrome
- 2018
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Ingår i: Iucrj. - : International Union of Crystallography (IUCr). - 2052-2525. ; 5:Part 5, s. 619-634
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Tidskriftsartikel (refereegranskat)abstract
- 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 myxobacterium Stigmatella aurantiaca (SaBphP1) and reveals its role in the fruitingbody 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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| 2. |
- Edlund, Petra, et al.
(författare)
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The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
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| 3. |
- Berntsson, Oskar, 1989, et al.
(författare)
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Sequential conformational transitions and alpha-helical supercoiling regulate a sensor histidine kinase
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases.
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| 4. |
- Herukka, S. K., et al.
(författare)
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Amyloid-beta and Tau Dynamics in Human Brain Interstitial Fluid in Patients with Suspected Normal Pressure Hydrocephalus
- 2015
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Ingår i: Journal of Alzheimers Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 46:1, s. 261-269
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Tidskriftsartikel (refereegranskat)abstract
- Background: Amyloid-beta (A beta(1-42)), total tau (T-tau), and phosphorylated tau (P-tau(181)) in the cerebrospinal fluid (CSF) are the most promising biomarkers of Alzheimer's disease (AD). Still, little is known about the dynamics of these molecules in the living brain. In a transgenic mouse brain, soluble A beta decreases with increasing age and advanced A beta pathology as seen similarly in CSF. Objective: To assess the relationship between AD-related pathological changes in human brain tissue, ventricular and lumbar CSF, and brain interstitial fluid (ISF). Methods: Altogether 11 patients with suspected idiopathic normal pressure hydrocephalus underwent frontal cortical brain biopsy, 24-h intraventricular pressure monitoring, and a microdialysis procedure. AD-related biomarkers were analyzed from brain tissue, CSF, and ISF. Results: ISF T-tau levels decreased strongly within the first 12 h, then plateauing until the end of the experiment. A beta(1-42) and P-tau(181) remained stable during the experiment (n = 3). T-tau and P-tau were higher in the ISF than in ventricular or lumbar CSF, while A beta(1-42) levels were within similar range in both CSF and ISF samples. ISF P-tau correlated with the ventricular CSF T-tau (r = 0.70, p = 0.017) and P-tau(181) (r = 0.64, p = 0.034). Five patients with amyloid pathology in the brain biopsy tended to reveal lower ISF A beta(1-42) levels than those six without amyloid pathology. Conclusions: This is the first study to report ISF A beta and tau levels in the human brain without significant brain injury. The set-up used enables sampling from the brain ISF for at least 24 h without causing adverse effects due to the microdialysis procedure to follow the dynamics of the key molecules in AD pathogenesis in the living brain at various stages of the disease.
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| 5. |
- Björling, Alexander, 1983, et al.
(författare)
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Structural photoactivation of a full-length bacterial phytochrome
- 2016
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 2:8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Björling, Alexander, 1983, et al.
(författare)
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Ubiquitous Structural Signaling in Bacterial Phytochromes
- 2015
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Ingår i: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 6:17, s. 3379-3383
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Tidskriftsartikel (refereegranskat)abstract
- The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.
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| 7. |
- Takala, H., et al.
(författare)
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On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:21, s. 8161-8172
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr(263)) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr(263) hydroxyl destabilizes the -sheet conformation of the tongue. This allowed the phytochrome to adopt an -helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr(263) in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.
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| 8. |
- Berntsson, Oskar, 1989, et al.
(författare)
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Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions
- 2019
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:7
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Tidskriftsartikel (refereegranskat)abstract
- Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome.
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| 9. |
- Ihalainen, J. A., et al.
(författare)
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Chromophore-Protein Interplay during the Phytochrome Photocycle Revealed by Step-Scan FTIR Spectroscopy
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:39, s. 12396-12404
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Tidskriftsartikel (refereegranskat)abstract
- Phytochrome proteins regulate many photo-responses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply twocolor step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H2O and D2O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved "PHY-tongue" region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C=O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation.
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| 10. |
- Lenngren, N., et al.
(författare)
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Coordination of the biliverdin D-ring in bacteriophytochromes
- 2018
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 20:27, s. 18216-18225
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Tidskriftsartikel (refereegranskat)abstract
- Phytochrome proteins translate light into biochemical signals in plants, fungi and microorganisms. Light cues are absorbed by a bilin chromophore, leading to an isomerization and a rotation of the D-ring. This relays the signal to the protein matrix. A set of amino acids, which is conserved across the phytochrome superfamily, holds the chromophore in the binding pocket. However, the functional role of many of these amino acids is not yet understood. Here, we investigate the hydrogen bonding network which surrounds the D-ring of the chromophore in the resting (Pr) state. We use UV/vis spectroscopy, infrared absorption spectroscopy and X-ray crystallography to compare the photosensory domains from Deinococcus radiodurans, the phytochrome 1 from Stigmatella aurantiaca, and a D. radiodurans H290T mutant. In the latter two, an otherwise conserved histidine next to the D-ring is replaced by a threonine. Our infrared absorption data indicate that the carbonyl of the D-ring is more strongly coordinated by hydrogen bonds when the histidine is missing. This is in apparent contrast with the crystal structure of the PAS-GAF domain of phytochrome 1 from S. aurantiaca (pdb code 4RPW), which did not resolve any obvious binding partners for the D-ring carbonyl. We present a new crystal structure of the H290T mutant of the PAS-GAF from D. radiodurans phytochrome. The 1.4 A-resolution structure reveals additional water molecules, which fill the void created by the mutation. Two of the waters are significantly disordered, suggesting that flexibility might be important for the photoconversion. Finally, we report a spectral analysis which quantitatively explains why the histidine-less phytochromes do not reach equal Pfr-type absorption in the photoequilibrium compared to the Deinococcus radiodurans wild-type protein. The study highlights the importance of water molecules and the hydrogen bonding network around the chromophore for controlling the isomerization reaction and spectral properties of phytochromes.
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