1.
Illuminating the inner workings of a natural protein switch: Blue-light sensing in LOV-activated diguanylate cyclases.
Abstract:
Regulatory proteins play a crucial role in adaptation to environmental cues. Especially for lifestyle transitions, such as cell proliferation or apoptosis, switch-like characteristics are desirable. While nature frequently uses regulatory circuits to amplify or dampen signals, stand-alone protein switches are interesting for applications like biosensors, diagnostic tools, or optogenetics. However, such stand-alone systems frequently feature limited dynamic and operational ranges and suffer from slow response times. Here, we characterize a LOV-activated diguanylate cyclase (LadC) that offers precise temporal and spatial control of enzymatic activity with an exceptionally high dynamic range over four orders of magnitude. To establish this pronounced activation, the enzyme exhibits a two-stage activation process in which its activity is inhibited in the dark by caging its effector domains and stimulated upon illumination by the formation of an extended coiled-coil. These switch-like characteristics of the LadC system can be used to develop new optogenetic tools with tight regulation.
2.
Influence of the N-terminal segment and the PHY-tongue element on light-regulation in bacteriophytochromes.
Abstract:
Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes. Red light–sensing bacteriophytochromes are attractive targets for the development of innovative optogenetic tools because of their natural modularity of coupling with diverse functionalities and the natural availability of the light-absorbing biliverdin chromophore in animal tissues. However, a rational design of such tools is complicated by the poor understanding of molecular mechanisms of light signal transduction over long distances—from the site of photon absorption to the active site of downstream enzymatic effectors. Here we show how swapping structural elements between two bacteriophytochrome homologs provides additional insight into light signal integration and effector regulation, involving a fine-tuned interplay of important structural elements of the sensor, as well as the sensor–effector linker. Facilitated by the availability of structural information of inhibited and activated full-length structures of one of the two homologs (Idiomarina species A28L phytochrome-activated diguanylyl cyclase (IsPadC)) and characteristic differences in photoresponses of the two homologs, we identify an important cross-talk between the N-terminal segment, containing the covalent attachment site of the chromophore, and the PHY-tongue region. Moreover, we highlight how these elements influence the dynamic range of photoactivation and how activation can be improved to light/dark ratios of ∼800-fold by reducing basal dark-state activities at the same time as increasing conversion in the light state. This will enable future optimization of optogenetic tools aiming at a direct allosteric regulation of enzymatic effectors.
