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.1-9 Bacterial phytochromes consist of a photosensory core and a C-terminal regulatory domain.10,11 Structures of photosensory cores are reported in the resting state12-18 and conformational responses to light activation have been proposed in the vicinity of the chromophore.19-23 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 active states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures reveal an open and closed form of the dimeric protein for the signalling and resting state, 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 where atomic scale conformational changes around the chromophore are first amplified into an Ångström scale distance change in the tongue, and further grow into a nanometre scale conformational signal. The structural mechanism is a blueprint for understanding how the sensor proteins connect to the cellular signalling network.

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. [1][2][3][4][5][6][7][8][9] Bacterial phytochromes consist of a photosensory core and a C-terminal regulatory domain. 10 The most common domain architecture of the photosensory core of phytochromes in bacteria, plants and fungi is PAS-GAF-PHY (Per/Arndt/Sim-cGMP phosphodiesterase/adenyl cyclase/FhlA-phytochromespecific). 10 We use the bacteriophytochrome from D. radiodurans to investigate the structural dynamics of photoswitching in these domains. Fig. 1a shows that the PAS-GAF-PHY domains share the prototypical response of phytochromes to red/far-red illumination. Its biliverdin chromophore relaxes into the Pr (red-absorbing) state when kept in the dark or after exposure to far-red light. The majority of molecules transform into Pfr (far-red absorbing) state after red light exposure.
The dark relaxation from Pfr to Pr occurs in a matter of days, and the optical response of PAS-GAF-PHY is very similar to the full-length protein. 24 For the PAS-GAF construct, which lacks the PHY domain, the absorption spectra ( Fig.   1a) and dark reversion within minutes indicate an incomplete photocycle. 24 Time-resolved solution X-ray scattering reports on structural changes in protein reactions. 25,26 We used it here to characterize the structural dynamics of the phytochrome photocycle. In a first experiment, the phytochrome fragments were repeatedly switched between the Pr and Pfr states using laser flashes of 10 ms duration at 671 nm (Pr  Pfr) and 750 nm (Pfr  Pr). X-ray scattering intensities were recorded in between the laser flashes for approximately 1 s and exposures after far-red flashes were subtracted from those of exposures after red flashes (see Supplementary Information for details). These difference scattering curves, ΔS(q), encode the structural change of the protein during the Pr  Pfr transition. The recorded difference scattering from the complete photosensory core (PAS-GAF-PHY) shows a large oscillating signal at low angles q < 2 nm -1 (Fig. 1b, red line), indicating large structural changes on the nanometre scale.
Oscillations at higher q, which report on structural changes on smaller length scales, were also observed (Extended Data Figure 1e), but are not discussed further here. The laser-induced ΔS of the PAS-GAF-PHY construct was reproduced by a standard Small-Angle X-ray Scattering (SAXS) experiment with pre-illuminated samples (Fig. 1b, blue line). Time-resolved difference X-ray scattering data covering the micro-and millisecond time scales also reproduce these features when photoconversion is complete (delay time: 30 ms, Fig. 1b, black line). These data further establish that the discussed low-q features grow in with a time-constant of 4.3 ms (Fig. 1b, Extended Data Figure 1d), which coincides with the formation of the Pfr state as measured by optical absorption of the chromophore (1.0 ms, Extended Data Figure 1d).
When performing the same experiments on the PHY-less photosensory core (PAS-GAF), the oscillatory signal at low-q is absent (Fig. 1c). The observed oscillations in the PAS-GAF domains are much smaller in amplitude and confined to higher q ranges ( Fig. 1c and Extended Data Figure 1e), indicating rearrangements of amino acid side chains and smaller shifts of secondary structural elements. 26 These data show that quaternary structural changes occur only when the PHY domain is present. In the wild-type protein the PHY domain connects the chromophore binding domains PAS/GAF with the output kinase, making it likely that this movement is functionally relevant.
In order to examine the nature of these conformational changes and to find out how they arise, we crystallised the photosensory core in the dark (referred to as dark) and under periodic illumination at 655 nm (referred to as illuminated), which transforms a large fraction of the protein molecules into the Pfr state ( Fig.   1a and Extended Data for details). Absorption spectra of the dark crystals indicate the Pr state, while the illuminated crystals are Pfr-enriched (Extended Data Figure 3c). Omit maps indicate a change in the conformation of the chromophore between the two crystal forms (Extended Data Figure 3b).
Although the biliverdin conformation cannot be fit unambiguously to the electron density in the illuminated structure, the remainder of the electron density is homogeneous (Extended Data Figure 2b).
Our dark crystal structure was modelled from data up to 3.80 Å resolution (Fig.   2a). The observed domain arrangement is similar to previously reported structures of bacterial PAS-GAF and PAS-GAF-PHY protein fragments, [12][13][14][15]17 but the dimer arrangement differs to some of these structures. 13,17 The illuminated structure, modelled against data cut at 3.24 Å, shows dramatic differences compared to the dark structure (Fig. 2a). Firstly, the dimer adopts an open Y-like shape of the PHY domains. This is in contrast to all reported resting state structures, where the dimer is closed (Fig. 2a). 12,13,17,20 Secondly, the tongue of the PHY domains (residues 446-477) appears as an α-helix and a loop in the illuminated model and is a β-sheet in the dark model ( Fig. 2b and Extended Data Figure 2). These two folds have been observed separately in prototypical cyanobacterial 13,17 and non-canonical bacterial phytochromes, 12,20 respectively (Extended Data Figure 4). Our structures establish that the refolding of the tongue occurs within the same bacterial phytochrome, and suggest that this refolding is associated with opening of the dimer. The change in fold causes the length of the tongue to vary between the dark and illuminated structures (Fig.   2b), which is key for understanding the role of the tongue in this signal relay.
Before detailing this mechanism, it is important to test how well the crystal models represent the solution structures of the Pr and Pfr states. To this end, we performed structural refinement against the solution difference X-ray scattering data ( Fig. 1), which is a very sensitive indicator of conformational change. It has the advantage over absolute scattering profiles that experimental error and uncertainties arising from the choice of method for scattering calculations largely cancel out (Extended Data 5).
We generated candidate solution structures by running two sets of molecular dynamics (MD) simulations starting from the dark and illuminated crystal structures (see Supplementary Information for details). These are referred to as Pr and Pfr trajectories since they aim to model solution scattering data representing these states. Snapshots were recorded every 50 ps and the solution X-ray scattering was calculated from each. All pairs of Pr/Pfr candidate structures were then compared to the static difference X-ray scattering (Fig. 1b, Extended Data Fig. 7, see Supplementary Information for details). We selected the 100 Pr/Pfr pairs with best agreement to the data and consider the participating structures to be representative of the protein in solution. Considering all pairwise differences between these structures, a consistent set of 747 curves was generated and evaluated (Fig. 3a). The agreement of the model and experiment is excellent, and striking improvement is made compared to the crystal structures.
In order to verify the choice of solution structures based on difference scattering, these structures were cross-validated against absolute, population-corrected SAXS data as described in Supplementary Information (see also Extended Data The solution-structural models show that the PHY domains of opposing monomers come fairly close to each other in the Pr state but move apart by ~3 nm during the Pr  Pfr transition (Fig. 4). The Y shape assumed by the solution structures in the Pfr state is in qualitative agreement with a low-resolution envelope of a Rps. Palustris phytochrome determined by SAXS. 27 Our crystal structures capture the essence of this quaternary conformational change, albeit with differences in the amplitude and details of the motion. These differences may be caused by crystal contacts (Extended Data Figure 6). Nevertheless, the qualitative agreement with the solution structures lends strong support to that the dark and illuminated crystal structures reflect the relevant structural rearrangement between Pr and Pfr states. Thus we consider the refolding of the tongue observed in the crystal structures to be a direct consequence of illumination. From the crystal and solution structures the following mechanism for signal transduction through the photosensory core emerges. It is known that the rotation of the biliverdin D-ring causes atomic rearrangements in the chromophore binding pocket, including displacement of Asp207 and Tyr263. [19][20][21][22] Our crystal structures suggest that this controls the fold of the tongue of the PHY domain. Importantly, the tongue is shortened as a result of the refolding and the distance between the GAF and PHY domain is reduced by 2.5 Å (Fig. 2b) upon red-light illumination. As a consequence of this, and demonstrated by our Pr and Pfr solution structures, the dimer opens up between the PHY domains by several nanometres (Fig. 4).
Three factors are essential for the proposed structural mechanism. Firstly, the PHY and GAF/PAS domains, which are connected by the tongue, are known to be very rigid internally such that they cannot deform to absorb the length variation of the tongue. 11 Secondly, the tongue and its junctions to PHY and GAF have to be rigid, especially in Pr where the PHY and GAF domains are pushed away from each other. In our dark structures the direction of the tongue is rigidly fixed with respect to the GAF domains by the conserved interaction Arg466 to Asp207/Tyr263 and additional backbone hydrogen bonding (Fig. 2b). 13 In the illuminated structure the tongue binds to GAF through interaction of Ser468 with Asp207/Tyr263, 12 and through hydrophobic interactions, among others by Tyr472 (Extended Data Fig. 4b). 17 The tongue is also locked close to the PHY domain by additional backbone hydrogen bonding. Thirdly, the PAS/GAF and PHY domains are connected by the unusually long scaffolding helix, which is necessary to redirect the shortening of the tongue into bending of the monomer.
Indeed, our simulations reveal a hinge in the scaffolding helix (Fig. 4b). All phytochrome structures published to date support these three requirements. [12][13][14][15][16][17][18] Phytochromes support different modes of signal output. In bacterial and fungal phytochromes output is through a C-terminal histidine kinase domain which autophosphorylates and further phosphotransfers to a response regulator. 3,7 In plant phytochromes two additional PAS domains are included in the C-terminal regulatory region and a more complex pattern of functions has to be controlled, such as serine/threonine kinase activity, 4 and affinity to interaction partners. 9,10 In all cases, the output activity is likely controlled by a structural change in the photosensory core. The photosensory core and the key amino acid sequence in the tongue region 465 PRxSF 469 are highly conserved over the whole phytochrome superfamily (Extended Data Figure 2c). We suggest here a mechanism, in which the shortening of the PHY-tongue pulls together (Pfr) or pushes apart (Pr) the PHY and PAS/GAF domains. The scaffolding helix redirects this force so that the monomers bend strongly. For the isolated photosensory unit in this study, this results in dramatic opening of the dimer ( Fig. 2 and Fig. 4). It is conceivable that full-length phytochromes modify the distance of output domains in a similar manner. Alternatively, previously buried patches of the protein could become accessible to interaction partners, or monomers could rearrange with respect to each other. It will be intriguing to see the emergence of these mechanisms, which become deductible with experimental approaches similar to the one presented here.

METHODS SUMMARY
The PAS-GAF-PHY and PAS-GAF domains from Deinococcus radiodurans were expressed in the Escherichia coli strain BL21 (DE3) and purified by affinity and size-exclusion chromatography. Crystallographic data was collected at beamline ID23-1 of the ESRF. Time-resolved X-ray scattering with millisecond time resolution were recorded at beamline cSAXS of the Swiss Light Source. 28

COMPETING FINANCIAL INTEREST
The authors declare no competing financial interest.

SUPPLEMENTARY INFORMATION
Supplementary materials and methods, Source Data, including solution structures, graphical data in ASCII format, sequence alignment, and force-field parameters for the biliverdin chromophore. shown. ΔS is the difference in scattered X-ray intensity caused by illumination at 671 nm. q = 4π/λ · sin θ at wavelength λ and scattering angle 2θ.   Note that the terms "illuminated" and "dark" refer here to the crystallization conditions (see Supplementary Information for details). The dark crystal spectrum resembles the Pr spectrum in solution (Fig. 1a). Illumination with red light in the crystallization drops at ambient temperature led to a slight increase of far-red absorption and disintegration of the crystals (data not shown). Dark crystals were unaffected by far-red illumination. The spectrum of the illuminated crystals shows that a substantial proportion (>50%) of the proteins reside in Pfr state. A similar absorption was detected from solubilized crystals.
The illuminated crystals could be switched to Pr-like absorption with far-red illumination. Reversely, also the Pfr-like features could be increased with red light (data not shown) with illumination at ambient temperature. Exposure with light increased the scattering background in the absorption measurements. The crystals seemed unaffected by the illumination when illuminated with red light in the crystallization drops.
Although the spectral analyses of the illuminated crystals do not indicate a pure Pfr spectrum, and the biliverdin conformation cannot be fit unambiguously to the electron density, the remainder of the electron density is homogeneous (Extended Data Figure   2b)   c-e, Consistency test of the structural refinement procedure. c, A cutoff parameter R cut was introduced to reject all MD frames R PP < R cut . The resulting average over R PP of the best N = 100 pairs is plotted as a function of R cut . It is found that R PP ≈ R cut , which indicates that the best fit to the difference X-ray scattering data is always at the highest separations available in sampling range. d and e show the dependence of the total and average error as a function of R cut , respectively. It is observed that the error decreases steeply for 5 nm, and only marginally for 5 nm. We therefore consider optimization in the latter range overfitting, and applied R cut = 5.0 nm in the refinement for the solution structures. The refined structures (Fig. 4) should therefore be considered to represent lower limits on R PP . f, Solution-structural refinement parameters and results.

Extended Data Table 1 | Crystallographic data collection and refinement statistics
Extended Data Table 2