Photocycle Structures of a Myxobacterial Phytochrome as Revealed by Cryo-EM

The ability of organisms to sense and respond to environmental stimuli is fundamental to survival. In bacteria, such responses are most commonly mediated by two-component signaling systems composed of a histidine kinase (HK) and a response regulator (RR) [1]. These systems are inherently modular, allowing sensory input domains to be structurally coupled to conserved signaling outputs. Many bacterial photoreceptors, including enzymatic light sensors, exploit this modularity by integrating photosensory domains into two-component signaling architectures, thereby converting changes in light spectrum, intensity, and direction into defined conformational and enzymatic responses. Phytochromes are among the best-characterized photoreceptors and are ubiquitous in plants as well as widespread in fungi and bacteria [2,3]. Despite sharing a conserved photosensory core, plant phytochromes (PHYs) differ in domain composition and likely in overall quaternary organization from their bacterial counterparts [4]. In plants, photoactivation induces large-scale structural rearrangements that promote nuclear translocation and downstream signaling events controlling germination, greening, and shade avoidance [5]. In contrast, bacterial phytochromes typically regulate enzymatic activity directly, often through coupling to two-component signaling pathways. In photosynthetic bacteria, phytochromes control the assembly of light-harvesting complexes [6,7], whereas in non-photosynthetic bacteria their structural basis for signaling remains poorly understood [8,9]. Recently, we investigated the structural and mechanistic basis of phytochrome signaling in the non-photosynthetic myxobacterium Stigmatella aurantiaca [9-11]. Myxobacteria are distinguished by a multicellular developmental program culminating in the formation of fruiting bodies. In S. aurantiaca, this process is regulated by red and far-red light, implicating bacteriophytochrome (BphP)-dependent signal transduction [9, 10, 12]. Understanding how light-induced structural changes in BphPs are transmitted to downstream effector domains is therefore central to elucidating light-controlled development in this organism. Beyond development, myxobacteria are prolific producers of secondary metabolites with anticancer and antimicrobial activity, whose biosynthesis has recently been shown to be modulated by light [13, 14], further underscoring the importance of phytochrome-mediated signaling. Bacteriophytochromes (BphPs) are multidomain proteins consisting of an N-terminal photosensory core module (PCM) and a C-terminal output effector module (OM) (Fig. 1 a). The PCM comprises three conserved domains—PAS (Per–Arndt–Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA), and PHY (phytochrome-specific GAF-related)—which together form a rigid yet photoresponsive scaffold. The bilin chromophore biliverdin (BV) (Fig. 1 b), a heme-derived open-chain tetrapyrrole composed of pyrrole rings A–D, is covalently attached to a conserved N-terminal cysteine and is deeply embedded within the GAF domain. Photoisomerization of BV triggers local rearrangements within the chromophore-binding pocket that propagate through the PCM, ultimately giving rise to global conformational changes. The C-terminal OM is covalently linked to the PHY domain and exhibits substantial variability among species, with histidine kinase domains being the most prevalent output modules [3,15,16]. Structural communication between the PCM and OM is believed to occur through light-induced reorganization of interdomain interfaces and changes in dimer geometry, but the molecular details of this coupling remain incompletely defined. In S. aurantiaca, two BphPs with similar domain architectures are present. In the classical bacteriophytochrome 2 (SaBphP2), the C-terminal OM is a histidine kinase (HK). SaBphP2 is encoded in a single operon together with genes encoding a cognate response regulator and a heme oxygenase required for biliverdin biosynthesis, providing a genetically compact system for signaling by the SaBphP2 photosensor. Cryo-EM structures of full length (intact) SaBphP2 particles including the HK domain were determined by us recently [17,18]. They provide detailed structural insight into a myxobacterial two-component signaling pathway in which light serves as the environmental input. This system operates in one of the most complex prokaryotic organisms, characterized by a multicellular life cycle and prolific production of secondary metabolites with antimicrobial and anticancer potential [14]. The reported cryo-EM structures resolve longstanding questions in phytochrome biology and overcome limitations that have constrained more than five decades of structural studies, which were largely restricted to fragmented proteins due to the difficulty of crystallizing intact photoreceptors. Comparative analysis of our cryo-EM structures supports a sequential, asymmetric mechanism for signal propagation from the photosensory core to the enzymatic output domain. Upon the absorption of a photon, the biliverdin (BV) chromophore in one protomer (subunit B) can undergo Z-to-E isomerization. Rotation of the BV D-ring induces substantial rearrangements within the chromophore-binding pocket, which in turn trigger a conformational transition of the sensory tongue in this protomer (Fig. 2 a). This local structural change is followed by moderate reorientations of the domains in the photosensory module and the appearance of a kink in the linker helix of the other, non-isomerized protomer (subunit A) (Fig. 2 a). Once a second photon is absorbed by subunit A, its sensory tongue also transitions to an α-helix. As a result, the coiled-coil linker of the protomer that isomerized first (subunit B) also displays a kink [17] (Fig. 2 b). Ultimately, the kinks in both linker helices propagate the signal to the C-terminal output module, which cause a rotation of the HK by about 60o. This results in an all-parallel configuration of the linker helices (Fig. 2 b). The rotation of the HK is likely to modulate the accessibility of the response regulator (RR) binding interface. An X-ray structure of an unrelated HK–RR complex demonstrates that RR binding is mediated primarily by the four-helix bundle of the HK [19]. Any structural perturbation of this four-helix bundle would therefore be expected to directly affect RR binding affinity and geometry. Accordingly, the light-induced rotation of the HK observed in SaBphP2 is poised to play a central role in controlling RR association and dissociation, and thus in regulating downstream signaling activity. Architecture and photocycle of SaBphP2. (a) Domain architecture assigned to sequence numbers. The biliverdin (BV), the sensory tongue and the coiled-coil linker helices are marked. (b) The photocycle of SaBphP2 with several intermediates. Upper half cycle: excited by red light. Lower half cycle: excited by far-red light. Inset: chemical structure of BV. Blue arrow: BV ring-D isomerization. Cryo-EM structures of SaBphP2. (a) The SaBphP2 Pr/Pfr heterodimer. PfrB: protomer B has undergone isomerization and is in the Pfr state, PrA: protomer A is not excited, and remains in the Pr state. The sensory tongues are marked by red spheres for protomers A (STA) and B (STB). The positions of the BVs are marked by dashed circles. The kink in the protomer that is not excited (still in Pr) is marked by an arrow. The HK lobes (HKA and HKB) are located on top of their respective opposite protomers. (b) Kink formation in the Pr/Pfr heterodimer and Pfr/Pfr homodimer. The PHY domains and the HK lobes are marked. The linker helices are marked with arrows. The kinks in the linker helices are marked for both states.