The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics.DOI: http://dx.doi.org/10.7554/eLife.11860.001

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the organism for using light energy. Illumination, however, enhances repression under semi-aerobic conditions. Here, we describe molecular details of two proteins involved in oxygen- and light-control of photosynthesis gene expression, the light-sensing anti-repressor AppA and the transcriptional repressor PpsR. We combine information from crystal structures of both proteins and their complex with hydrogen-deuterium exchange data to show that light-activation of AppA–PpsR2 affects the PpsR effector region within the complex. DNA-binding studies demonstrate the formation of a light-sensitive ternary AppA–PpsR–DNA complex. Implications of these results for light- and oxygen-regulation are discussed, highlighting new insights into blue-light-mediated signal transduction.

Organisms adapt to environmental cues using diverse signaling networks. In order to sense and integrate light for regulating various biological functions, photoreceptor proteins have evolved in a modular way. This modularity is targeted in the development of optogenetic tools enabling the control of cellular events with high spatiotemporal precision. However, the limited understanding of signaling mechanisms impedes the rational design of innovative photoreceptor-effector couples. Here, we reveal molecular details of signal transduction in phytochrome-regulated diguanylyl cyclases. Asymmetric structural changes of the full-length homodimer result in a functional heterodimer featuring two different photoactivation states. Structural changes around the cofactors result in a quasi-translational rearrangement of the distant coiled-coil sensor-effector linker. Eventually, this regulates enzymatic activity by modulating the dimer interface of the output domains. Considering the importance of phytochrome heterodimerization in plant signaling, our mechanistic details of asymmetric photoactivation in a bacterial system reveal novel aspects of the evolutionary adaptation of phytochromes.

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Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 μg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.