Abstract
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.
Highlights
Our previous characterization of the IsPadC system revealed that the phytochrome – diguanylyl cyclase (DGC) linker region populates two different coiled-coil register conformations that influence the enzymatic activity of the effector (Gourinchas et al, 2017)
All three constructs were expressed in an E. coli strain coexpressing heme oxygenase to isolate holoproteins loaded with the biliverdin co-factor and to enable a more detailed biochemical characterization
In the IsPadC (Gourinchas et al, 2017) system (Figure 1a), the bacteriophytochrome module is linked to a diguanylyl cyclase (DGC) effector (Ryjenkov et al, 2005) and controls formation of the bacterial second messenger cyclic-dimeric-GMP (Romling et al, 2013) by red light (Figure 1a–b)
Summary
Screening and expression of IsPadC variants stabilized in different coiled-coil registers. Comparing just the differences in deuterium incorporation between light- and dark-state conditions for the two systems reveals no major differences For both the wild-type and the IsPadCReg variant we observed an increase in conformational dynamics in the PHY-tongue region, the central helical spine and the coiled-coil sensor– effector linker upon illumination (Figure 4). A closer inspection of the deuterium exchange kinetics, revealed that the conformational dynamics of IsPadCReg, compared to the wild-type protein in either the dark-adapted state or during constant red light illumination, are reduced in several functionally important regions This is especially true for the PHY-tongue region, the coiled-coil and a GGDEF element close to the GTP binding site (Figure 4 and Figure 4—figure supplement 1). These broad distributions are indications for bimodal deuterium uptake that originates from two molecular species with different deuterium exchange kinetics, which would be in line with two biliverdin environments coupled to different PHYtongue conformations under steady-state light conditions
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