Light is a key stimulus for plant biological functions, several of which are controlled by lightactivated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, Light-Oxygen-Voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internallybound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jα helix, and ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue (Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain) switches its hydrogen-bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Iβ strand that interacts with the Jα helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jα helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudo-dark state (Q513L) or a pseudo-lit state (Q513N) conformation. Further, both mutations changed the photochemical properties of this receptor, particularly the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Iβ strand to the peripheral Jα helix.Protein signaling cascades are central to organismal growth, adaptation, and communication; therefore, the regulation of these cascades is key to survival. PAS (Per-ARNT-Sim) domaincontaining proteins are well characterized as vital members of many such regulatory paths, including adaptation to hypoxia (1), circadian rhythm-dependent gene transcription (2), and phototropism and chloroplast organization in plants (3). A specific subset of PAS proteins, the LOV (light-oxygen-voltage) domains (4), is capable of sensing blue light as an environmental signal and converting it into a biochemical signal in a wide variety of proteins.LOV domains contain a series of highly conserved residues surrounding an internally bound flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) chromophore (Fig. 1a, 1b) The sensory role played by LOV domains is characterized in a variety of proteins, including transcription factors, ubiquitin ligases and kinases. Previous studies on photo...
Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.two-component system | cell signaling | histidine kinase | photosensory | regulation
BlrB in Rhodobacter sphaeroides is a single domain, flavin-based blue light sensor protein in the BLUF family of photoreceptors. Consistent with other members of this family, blue light excitation induces a putative signaling state characterized by a 10 nm red shift in the UV-visible absorbance spectrum. Structural and spectroscopic characterization of truncated BlrB constructs establishes that the C-terminal 50 amino acids of this protein are essential to its structural integrity despite not being part of the canonical BLUF domain architecture. Mutagenesis studies support the critical roles of Tyr9, Asn33, and Gln51 for flavin binding and the integrity of the BLUF domain fold. Comparison of solution NMR spectra of BlrB acquired under dark and light conditions indicates very limited light-dependent conformational changes except for a few interesting residues: Trp92, Met94, and Ile127. Notably, the Ile127 side chain experiences significant chemical shift changes despite the fact that it is far ( approximately 15 A) from the flavin chromophore in the C-terminal extension. These data suggest that the light-induced signal is propagated from the flavin through the beta sheet to the last two alpha helices in the C-terminal extension, potentially providing a mechanism to transmit this change to initiate a cellular response to blue light.
Light is an essential environmental cue for diverse organisms. Many prokaryotic blue light photoreceptors use Light, Oxygen, Voltage (LOV) sensory domains to control the activities of diverse output domains, including histidine kinases (HK). Upon activation, these proteins autophosphorylate a histidine residue before subsequently transferring the phosphate to an aspartate residue in the receiver domain of a cognate response regulator (RR). Such phosphorylation, activates the output domain of the RR, leading to changes in gene expression, protein-protein interactions or enzymatic activities. Here we focus on one such light sensing LOV-HK from the marine bacterium Erythrobacter litoralis HTCC2594 (EL368), seeking to understand how kinase activity and subsequent downstream effects are regulated by light. We found that photoactivation of EL368 led to a significant enhancement in the incorporation of phosphate within the HK domain. Further enzymatic studies showed that the LOV domain affected both the LOV-HK turnover rate (kcat) and Km in a light-dependent manner. Using in vitro phosphotransfer profiling, we identified two target RRs for EL368 and two additional LOV-HKs (EL346 and EL362) encoded within the host genome. The two RRs include a PhyR-type transcriptional regulator (EL_PhyR) and a receiver-only protein (EL_LovR), reminiscent of stress-triggered systems in other bacteria. Taken together, our data provide a biochemical foundation for this light-regulated signaling module of sensors, effectors and regulators that control bacterial responses to environmental conditions.
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