Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Structural mechanism of signal transduction in a phytochrome histidine kinase.

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.

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 Šresolution 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 Šresolution 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.

High-resolution crystal structures of transient intermediates in the phytochrome photocycle.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.

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.

Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction. To detect the level of light, plant cells use proteins called phytochromes, which are also found in some bacteria and fungi. Phytochrome proteins change shape when they are exposed to red light, and this change alters the behaviour of the cell. The red light is absorbed by a molecule known as chromophore, which is connected to a region of the phytochrome called the PHY-tongue. This region undergoes one of the key structural changes that occur when the phytochrome protein absorbs light, turning from a flat sheet into a helix. Claesson, Wahlgren, Takala et al. studied the structure of a bacterial phytochrome protein almost immediately after shining a very brief flash of red light using a laser. The experiments revealed that the structure of the protein begins to change within a trillionth of a second: specifically, the chromophore twists, which disrupts its attachment to the protein, freeing the protein to change shape. Claesson, Wahlgren, Takala et al. note that this structure is likely a very short-lived intermediate state, which however triggers more changes in the overall shape change of the protein. One feature of the rearrangement is the disappearance of a particular water molecule. This molecule can be found at the core of many different phytochrome structures and interacts with several parts of the chromophore and the phytochrome protein. It is unclear why the water molecule is lost, but given how quickly this happens after the red light is applied it is likely that this disappearance is an integral part of the reshaping process. Together these events disrupt the interactions between the chromophore and the PHY-tongue, enabling the PHY-tongue to change shape and alter the structure of the phytochrome protein. Understanding and controlling this process could allow scientists to alter growth patterns in plants, such as crops or weeds.

High-resolution crystal structures of a myxobacterial phytochrome at cryo and room temperatures.

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.

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity.

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter.

Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site.

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.

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 myxo-bacterium Stigmatella 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.

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.

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 Å 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 Å 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.