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.
Highlights
Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes
The photosensory modules (PSMs)2 of bacteriophytochromes are usually composed of a period/ARNT/single-minded (PAS) domain preceded by a N-terminal segment (NTS) that covalently binds the open-chain tetrapyrrole biliverdin chromophore via a thioether linkage with a conserved cysteine residue [9], a cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain stabilizing the chromophore by polar and hydrophobic interactions, and a phytochrome-specific (PHY) domain stabilizing the photoactivated Pfr state [10, 11]
Considering PadCs as powerful model systems for phytochrome function, where structural studies have recently improved the understanding of molecular details involved in the long-range signal transduction mechanisms of phytochromes [15, 36], we addressed the involvement and cross-talk between characteristic structural elements in bacteriophytochrome signaling, the NTS, and the PHY-tongue
Summary
IsPadC and TsPadC share the same domain organization and highly conserved residues around the biliverdin-binding pocket (Fig. 1). Recording the dark and illuminated spectra of TsPadC at low temperature, to minimize thermal reversion processes, resulted in increased Pfr-like contributions at 744 nm accompanied by a shift of the isosbestic point of the Pr and illuminated photoequilibrium spectra (Fig. 2C). This indicates the presence of more than two species contributing to the steady-state illuminated spectra of TsPadC. The biliverdin chromophore in ZZEssa configuration, comparable with the symmetrically activated Deinococcus radiodurans bacteriophytochrome (DrBphP) featuring a classical Pfr spectrum of both protomers [11, 13] (Fig. 2B) These observations support that both IsPadC and TsPadC feature heteroge-. IsPadC [36] IsPadCϩ7KE IsN/PG/Yt/Y/CCTsD Is Ts N/PG/Yt/Y/CC⌬515–521 D Is Ts N/PG/Yt/Y/CC501–507 CC505–518/D IsN/PG/Yt/YTsCC/D IsN/PGTsYt/Y/CC/D TsNIsPG/Yt/Y/CCTsD TsNIsPGTsYt/Y/CC/D TsNIsPGTsYtIsY/CC/D TsN/PG/Yt/YIsCC/D TsN/PG/Yt/Y/CCIsD
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