Signal Transduction in an Enzymatic Photoreceptor Revealed by Cryo-Electron Microscopy

Abstract

Phytochromes are essential photoreceptor proteins in plants with homologs in bacteria and fungi that regulate a variety of important environmental responses. They display a reversible photocycle between two distinct states, the red-light absorbing Pr and the far-red light absorbing Pfr, each with its own structure. The reversible Pr to Pfr photoconversion requires covalently bound bilin chromophore and regulates the activity of a C-terminal enzymatic domain, which is usually a histidine kinase (HK). In plants, phytochromes translocate to nucleus where the C-terminal effector domain interacts with protein interaction factors (PIFs) to induce gene expression. In bacteria, the HK phosphorylates a response-regulator (RR) protein triggering downstream gene expression through a two-component signaling pathway. Although plant and bacterial phytochromes share similar structural composition, they have contrasting activity in the presence of light with most BphPs being active in the dark. The molecular mechanism that explains bacterial and plant phytochrome signaling has not been well understood due to limited structures of full-length phytochromes with enzymatic domain resolved at or near atomic resolution in both Pr and Pfr states. Here, we report the first Cryo-EM structures of a wild-type bacterial phytochrome with a HK enzymatic domain, determined in both Pr and Pfr states, between 3.75 and 4.13 Å resolution, respectively. Furthermore, we capture a distinct Pr/Pfr heterodimer of the same protein as potential signal transduction intermediate at 3.75 Å resolution. Our three Cryo-EM structures of the distinct signaling states of BphPs are further reinforced by Cryo-EM structures of the truncated PCM of the same protein determined for the Pr/Pfr heterodimer as well as Pfr state. These structures provide insight into the different light-signaling mechanisms that could explain how bacteria and plants see the light.

Dynamics-driven allosteric stimulation of diguanylate cyclase activity in a red light-regulated phytochrome

“…In the context of long-range signal integration, this observation supports mixed Pfr/intermediate-state and apparently also Pfr/Pr “hetero”-dimers as functional units in red light activation of PadCs ( 24 ). Since recently Pr/Pfr heterodimers were also structurally observed for Stigmatella aurantiaca phytochrome 1 ( Sa BphP1, ( 48 )) this feature could be more generally relevant in the field of bacteriophytochromes than initially expected. In the context of plant phytochromes, phyA Pr/Pfr heterodimers are probably responsible for the very low fluence response ( 49 ) and also phyB Pr/Pfr dimers feature distinct biophysical properties with functional implications for plant morphogenic development ( 50 ).…”

“…This results in the population of different coiled-coil registers, one of which stimulates DGC activity as recently also seen for other GGDEF domains ( 54 ). In the overall context of phytochromes, the appreciation of dynamics-driven allostery complements insights into effector activation obtained by other approaches, like NMR ( 55 ), crystallography ( 24 ), or cryo-EM ( 48 , 56 ).…”

Cryo-EM structures of a bathy phytochrome histidine kinase reveal a unique light-dependent activation mechanism

“…The open structure is surprising in light of the two recently solved full-length structures of Pfr-state bacteriophytochromes, which showed a closed arrangement of the photosensory module dimer 30,31 . The missing densities in this dataset could in theory be explained by compositional heterogeneity.…”

“…The loops reveal that the phosphorylation reaction will occur in between the CA domain of protomer A and catalytic histidine of protomer B (and vice versa ), which is known as trans -autophosphorylation (Fig. S5) 30,31 . The orientation of the domains suggests a kinase-active state 52 .…”

“…This group contains a number of other phytochromes from non-photosynthetic bacteria. Although the constitution of the dimer interface in the neck region will also play a role, the absence of 10 residues is a probable explanation to why Pa BphP opens up in Pfr while Dr BphP and Sa BphP2 do not 30,31 .…”