Cryo-electron microscopy of Arabidopsis thaliana phytochrome A in its Pr state reveals head-to-head homodimeric architecture

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM

Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics. The bacteriophytochrome DrBphP from Deinococcus radiodurans shows high sequence homology to the histidine kinase Agp1 from Agrobacterium fabrum but lacks kinase activity. Here, the authors structurally and biochemically analyse DrBphP and Agp1, showing that DrBphP is a light-activatable phosphatase.Peer reviewe

The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature

(6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 angstrom resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 angstrom resolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.Peer reviewe

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.Peer reviewe

Structural Insights at Sub-Ångstrom, Medium and Low Resolution: Crystallization of Trypsin, Bacterioferritin, Photosynthetic Reaction Center, and Photosynthetic Core Complex

The catalytic action of serine proteases depends on the interplay of a nucleophile, a general base and a general acid. The catalytic triad is composed of serine, histidine and aspartate residues. The serine acts as a nucleophile while the histidine plays a dual role as the general base or acid at different steps of the reaction. However, the role of aspartate is unclear. I recovered an ultrahigh resolution (0.93 Å) X-ray structure of a complex formed between trypsin and a canonical inhibitor. At sub-ångstrom resolution, hydrogen atoms could be visualized, giving a clue to the protonation state of the catalytic residues. By comparing this with the theoretical electron density calculated by density theory functional, the protonation states of the catalytic histidine and aspartate are discussed. Hence, a refined mechanism for serine protease action is proposed in this thesis. Photosystem harvests energy from sunlight with near 100% quantum yield. To study light-induced structural changes of the photosynthetic reaction center from purple non-sulfur bacterium Blastochloris viridis using X-ray crystallography, robust protein crystals with tight crystal packing are prerequisite. In this thesis, lipidic-sponge phase crystallization method was used and yielded well diffracting crystals for structure determination. Crystals showed a type I packing and a 1.86 Å resolution structure was determined with four lipid molecules captured in the structure. Moreover, I demonstrated that an occupied QB binding site can be obtained by co-crystallizing with UQ2 using the sponge phase crystallization method. However, attempting to crystallize the reaction center-light harvesting 1 core complex, a 440 kDa membrane protein complex of total 54 putative subunits, it required different crystallization methods. Here, the resolution has been optimized to beyond 8 Å by using the lipidic bicelle crystallization method. Conflict between the free but potential toxic Fe(II) and the insolubility of Fe(III) led to the evolution of bacterioferritin in bacteria, which functions as an iron storage and detoxification protein. Bacterioferritin from Blastochloris viridis (Bv Bfr) was crystallized and the structure was solved to 1.58 Å resolution. With the combination of X-ray structure, redundancy PCR and tandem mass spectrometry, the previously unknown amino acid sequence of Bv Bfr was determined. Conformational states of the ferroxidase center which undergoes reorganization upon different soakings were trapped. One water-like small ligand coordinated to the Fe1 binding site was captured in the Fe(II)-soaked structure. By density functional theory calculations the character of this small legend was rationalized. In addition, the structure and mechanism of iron import of the protein was studied and discussed. Finally, the redox-state of the heme in the crystals with and without Fe(II)-soaking treatment was studied by single crystal UV-VIS microspectrophotometry, before and after the X-ray exposure

Lipidic sponge phase crystal structure of a photosynthetic reaction center reveals lipids on the protein surface.

Membrane proteins are embedded in a lipid bilayer and maintain strong interactions with lipid molecules. Tightly bound lipids are responsible for vertical positioning and integration of proteins in the membrane and for assembly of multisubunit complexes and occasionally act as substrates. In this work we present the lipidic sponge phase crystal structure of the reaction center from Blastochloris viridis to 1.86 A, which reveals lipid molecules interacting with the protein surface. A diacylglycerol molecule is bound, through a thioether bond, to the N-terminus of the tetraheme cytochrome c subunit. From the electron density recovered at the Q(B) site and the observed change in recombination kinetics in lipidic sponge phase-grown crystals, the mobile ubiquinone appears to be displaced by a monoolein molecule. A 36 A long electron density feature is observed at the interface of transmembrane helices belonging to the H- and M-subunits, probably arising from an unidentified lipid

Structural mechanism of signal transduction in a phytochrome histidine kinase

Publisher Copyright: © 2022, The Author(s).Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.Peer reviewe

Structural mechanism of signal transduction in a phytochrome histidine kinase

Publisher Copyright: © 2022, The Author(s).Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.Peer reviewe

Kinetic, Thermodynamic, and Crystallographic Studies of 2-Triazolylthioacetamides as Verona Integron-Encoded Metallo-β-Lactamase 2 (VIM-2) Inhibitor

Inhibition of β-lactamases presents a promising strategy to restore the β-lactams antibacterial activity to resistant bacteria. In this work, we found that aromatic carboxyl substituted 2-triazolylthioacetamides 1a–j inhibited VIM-2, exhibiting an IC50 value in the range of 20.6–58.6 μM. The structure-activity relationship study revealed that replacing the aliphatic carboxylic acid with aromatic carboxyl improved the inhibitory activity of 2-triazolylthioacetamides against VIM-2. 1a–j (16 mg/mL) restored the antibacterial activity of cefazolin against E. coli cell expressing VIM-2, resulting in a 4–8-fold reduction in MICs. The isothermal titration calorimetry (ITC) characterization suggested that the primary binding 2-triazolylthioacetamide (1b, 1c, or 1h) to VIM-2 was a combination of entropy and enthalpy contributions. Further, the crystal structure of VIM-2 in complex with 1b was obtained by co-crystallization with a hanging-drop vapour-diffusion method. The crystal structure analysis revealed that 1b bound to two Zn(II) ions of the enzyme active sites, formed H-bound with Asn233 and structure water molecule, and interacted with the hydrophobic pocket of enzyme activity center utilizing hydrophobic moieties; especially for the phenyl of aromatic carboxyl which formed π-π stacking with active residue His263. These studies confirmed that aromatic carboxyl substituted 2-triazolylthioacetamides are the potent VIM-2 inhibitors scaffold and provided help to further optimize 2-triazolylthioacetamides as VIM-2 even or broad-spectrum MβLs inhibitors

Applying THz Technology in Life Science

Bio-molecules, due to their structure and size, havevibrational and rotational resonances at THz frequencies(0.1 to 10 THz) [1]. This unique interactionpromises a multitude of applications in life sciences,wherein the latest development of THz sources anddetectors has enabled this field of research to grow.Although being at an early and mostly exploratorystage of development, this field is riddled with manychallenges that should be overcome; measurementsof inhomogeneous media, short interaction lengthswith few molecules, strong water absorption, and alsothe cultural gap between medical and engineeringcommunities.In this work we present our preliminary results forthree experiments within this field