A tour de force of X-ray scattering has yielded structures of a phytochrome photoreceptor in its dark and illuminated states, showing how localized protein refolding magnifies a light signal to form a cellular message. See Letter p.245
Sensor proteins allow organisms to perceive and appropriately respond to environmental changes. They are molecular machines comprised of sensory domains and effector modules; a specific signal is received by the former and communicated to the latter through structural changes, creating an output activity that directs a cellular response. Phytochromes are dimeric sensor proteins that specifically absorb red and near-infrared light using a covalently tethered chromophore molecule housed in a light-sensitive (photosensory) core. In this issue, Takala et al.1 (page 245) demonstrate how visible light is interpreted and spatially magnified by the phytochrome dimer in a chain reaction that uses key features of the protein's three-dimensional structure.
Phytochromes regulate most of the responses of plants to light, including germination, flowering and shade avoidance, and are thus fundamental to agriculture2. They are also widespread in microbes3, and phytochromes found in cyanobacteria are thought to be ancestors of those in plants2. The proteins signal by switching between two stable conformations, which correspond to the dark and illuminated states. Absorption of light by the chromophore alters a local network of hydrogen bonds and van der Waals interactions, and ultimately induces the illuminated conformation.
How these minute changes (of the order of ångströms) are transferred through the protein to regulate the distant effector domain is a long-standing puzzle. One proposed model, also applicable to blue-light receptors and sensors that react to chemical signals, involves the rotation of central helices connecting the sensory and effector modules4. In phytochromes, a structural element known as the tongue also spans the two modules and is likewise expected to be involved in signal transduction5.
The inherent dynamism of the dark and illuminated states confounds structural studies of phytochromes. Several dark-state crystal structures of domains of the photosensory core have been solved6,7, but finding a way to consistently trap the phytochrome in the illuminated state for crystallography has been a major obstacle. Takala and colleagues overcame this problem by taking advantage of the fact that the phytochrome from the bacterium Deinococcus radiodurans relaxes slowly in vitro from its illuminated state to its dark conformation. The authors therefore delivered periodic red-light pulses to growing crystals of the protein, to enrich the population of molecules in the illuminated state, and thereby enable a stable crystal of the sensor in that conformation to be grown. This allowed low-resolution X-ray structures to be solved for the complete photosensory core in the dark and illuminated crystalline states.
Next, the researchers performed molecular dynamics simulations based on the crystal structures to generate structures of the protein in solution. These were validated by comparison with X-ray-scattering data for the photosensory core in solution. Together, the crystal and solution structures support a model in which chromophore isomerization resulting from red-light absorption ruptures adjacent hydrogen bonds, causing a β-hairpin structural feature in the tongue to unfold and nearby atoms in the same malleable structural unit to wind into an α-helix. The tongue thus contracts by 2.5 Å to relieve a sharp (approximately 50°) bend at a hinge in the central helix. The resulting illuminated state has a distinct Y shape, in which the arms of the Y are as much as 8 nanometres apart. In an experimental tour de force, the authors combined time-resolved X-ray-scattering data collected over several timescales in solution, and found that the phytochrome magnifies light-induced atomic-scale changes (produced within picoseconds6; 1 picosecond is 10−12 s) to the nanometre scale in milliseconds (Fig. 1).
Models of signal transduction in which the central helix of the phytochrome rotates are not supported by these findings, but are supplanted by a scenario in which the helix straightens in response to refolding of the tongue when illuminated. This explains why two tongue conformations have previously been seen for phytochromes6,7. The new model is supported by earlier observations8 of light-induced effects near the chromophore, and plausibly connects structural changes from the atomic to the nanometre scale. A central tenet of the mechanism is that the secondary structure of the tongue changes, but could it be that the observed conformations occur only in crystals, and not in solution? Such concerns are largely allayed by the triangulation of results obtained using molecular dynamics, solution scattering and protein crystallography.
The tongue-refolding model requires that dramatic movements within the protein are manifested through a plastic region in the central helix; for this to occur, kinetic energy must not be misdirected into random motion throughout the photosensory core. Phytochromes contain an unusual figure-of-eight knot9, and we propose that this limits the number of degrees of conformational freedom available to the protein when an energetic red-light photon is absorbed. This rigidity presumably funnels the protein's response to the appropriate changes in secondary structure along the chromophore–tongue–effector-domain trajectory so that signal transduction can proceed.
Takala and co-workers provide a picture of phytochrome signal transduction that suggests a mechanism for how the activity of the effector domain is regulated by red light. In D. radiodurans, the phytochrome effector module is a histidine kinase. In the illuminated state, the ends of the two Y arms each bear one of these enzymatic modules. We infer that the trajectory of the central helix observed in each monomer of the illuminated structures continues through the kinase domain, although this remains to be proven experimentally. In any case, strain induced by straightening of the long central helix will force the effector modules into a new orientation relative to one another.
Histidine kinases act by transferring a phosphate group to their substrate across a subunit–subunit interface10. In the case of the phytochrome, it is logical to conclude that enzymatic activity occurs in the dark, when the active site of an effector module is close to the phosphorylation site of its partner, but halts in the light, when the modules are separated11. Unfortunately, crystallization of dynamic histidine kinases suffers from similar roadblocks to those associated with phytochrome crystallization. A complete phytochrome structure at near-atomic resolution in both states therefore remains the ultimate goal in the field, along with accompanying enzymological and physiological studies to support any structural model that shows how local structural changes are magnified into global ones.
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Baker, A., Forest, K. Action at a distance in a light receptor. Nature 509, 174–175 (2014). https://doi.org/10.1038/nature13331
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