Oskar Berntsson

Signal amplification and transduction in phytochrome photosensors

Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain. Structures of photosensory cores are reported in the resting state and conformational responses to light activation have been proposed in the vicinity of the chromophore. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved ‘tongue’, which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

Structural photoactivation of a full-length bacterial phytochrome

Time-resolved x-ray solution scattering reveals the conformational signaling mechanism of a bacterial phytochrome. 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.

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.

Ubiquitous Structural Signaling in Bacterial Phytochromes

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.

Time-Resolved X-Ray Solution Scattering Reveals the Structural Photoactivation of a Light-Oxygen-Voltage Photoreceptor

Light-oxygen-voltage (LOV) receptors are sensory proteins controlling a wide range of organismal adaptations in multiple kingdoms of life. Because of their modular nature, LOV domains are also attractive for use as optogenetic actuators. A flavin chromophore absorbs blue light, forms a bond with a proximal cysteine residue, and induces changes in the surroundings. There is a gap of knowledge on how this initial signal is relayed further through the sensor to the effector module. To characterize these conformational changes, we apply time-resolved X-ray scattering to the homodimeric LOV domain from Bacillus subtilis YtvA. We observe a global structural change in the LOV dimer synchronous with the formation of the chromophore photoproduct state. Using molecular modeling, this change is identified as splaying apart and relative rotation of the two monomers, which leads to an increased separation at the anchoring site of the effector modules.

Sequential conformational transitions and α-helical supercoiling regulate a sensor histidine kinase

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.Sensor histidine kinases (SHK) consist of sensor, linker and kinase modules and different models for SHK signal transduction have been proposed. Here the authors present nano- to millisecond time-resolved X-ray scattering measurements, which reveal a structural mechanism for kinase domain activation in SHK.

Light-induced structural changes in a monomeric bacteriophytochrome

Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

Terahertz absorption of illuminated photosynthetic reaction center solution: a signature of photoactivation?

Photosynthetic reaction centers develop a stable charge separated state upon illumination. To investigate the molecular vibrations associated with the illuminated state of a reaction center we recorded terahertz absorption spectra of the photosynthetic reaction center from Rhodobacter sphaeroides in the dark and upon illumination and observed a small, but significant THz absorption increase in the 20 to 130 cm−1 spectral region. Reaction centers show very similar terahertz absorption increase when solubilized in detergents and in a lipidic sponge phase indicating that the nature of the bulk solvent has limited influence on the vibrational spectrum. The absorption change of the isolated LM subunit is very similar to that of the intact reaction center. Through temperature control experiments we show that 89% of the absorption change is likely attributed to the non-thermal activation of the protein molecules. These results indicate that picosecond molecular vibrations change primarily in the cofactors and/or in the evolutionary conserved core of the reaction center upon illumination, whereas the nuclear motions of the H-subunit and the bulk solvent have limited impact on the terahertz spectral changes.