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.

Leukocyte integrins of the ␤2 family are essential for immune cell-cell adhesion. In activated cells, ␤2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the ␤2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of ␤2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated ␤2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K d , 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K d , 0.5 mM). Phosphorylation did not regulate talin binding to ␤2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated ␤2 integrin. X-ray crystallographic data clearly IntroductionIntegrins are heterodimeric plasma membrane receptors that mediate binding to the extracellular matrix and to ligands present on the surface of other cells. Their function is tightly regulated; they bind ligands only after activation. Modulation of integrin activity occurs through tightly regulated interactions between cytoplasmic molecules and integrin intracellular tails. Factors binding to integrin cytoplasmic domains regulating integrin adhesiveness include the cytoskeletal proteins talin 1,2 and filamin, 3 and the 14-3-3 proteins, which are molecular adaptors that bind to phosphorylated serine or threonine (pSer/ pThr) containing polypeptide sequences. 4 The ␤2 integrins are expressed exclusively on leukocytes and bind ICAM molecules on other leukocytes and endothelial cells after cell activation. 5,6 Talin binds to ␤2 integrins in vitro and in cells and is involved in activating the ␤2 integrins, resulting in binding to ICAMs. 1,4,[7][8][9] The ␤2 integrin polypeptide chain is phosphorylated on the intracellular domain on several residues after cell stimulation with various agents. 10 Thr758 is a physiologically important amino acid residue in the ␤2 cytoplasmic tail, and becomes phosphorylated after T-cell stimulation with T-cell receptor (TCR) antibodies or with phorbol esters. [11][12][13] After its phosphorylation, ␤2 binds to 14-3-3 proteins both in vitro and in cells. 4 Blocking of this interaction with a ␤2 Thr758 to Ala mutation, or by expression of constructs that bind to 14-3-3 proteins and block their interactions with target proteins, leads to abrogation of actin cytoskeleton rearrangements, cell spreading, and adhesion to ICAM ligands. 4 ␤2-Thr758 phosphorylation leads to the activation of the actin cytoskeleton modulators, Rac1/Cdc42, in cells. 13 The region in the ␤2 cytoplasmic tail that binds 14-3-3 proteins has been reported to interact with filamin in other integrins, 14 and for the strong filamin-binder ␤7 integrin, phosphorylation mimicking substitutions of 3 threonine residues (TTT) reduces filamin affinity. 3 Fi...

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

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.

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 PRSF 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) in the phytochrome from the extremophile bacterium 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 hydroxyl destabilizes the β-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-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 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.

Background: Bacteriophytochromes are dimeric histidine kinases, but the functional role of their dimerization interfaces is unclear. Results: The phytochrome from Deinococcus radiodurans has two dimerization interfaces, which are critical for thermal back reversion and are altered by illumination. Conclusion:The dimerization interfaces cause strain in the structure. Significance: A functional role for the dimerization interfaces is proposed.

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.

Light control of cell development is revealed by phytochrome structures of Myxobacteria.