Signal Integration in Phytochrome-linked Diguanylyl Cyclases

During evolution, nature has developed an astonishingly modular architecture of directly coupled individual protein domains. Using an array of building blocks with diverse functionalities, such as environmental sensor domains, interaction modules or enzymatic output units, enabled organisms to develop complex cellular networks that are critical for cell survival. The frequently observed linkage of sensory modules with enzymatic effectors enables direct regulation of, for example, the level of important signalling molecules for adapting the lifestyle of an organism in response to diverse environmental stimuli. The interest in light-regulated systems has recently increased due to the establishment of optogenetics, which refers to the concept of genetically targeting biological systems to enable optical control of cellular processes. However, the demand for light-controlled systems goes beyond that of naturally occurring photoreceptors. Even though progress in understanding concepts of light activation has been made, the rational design of synthetic tools is still challenging. Since mechanistic descriptions of light-signalling differ even within photoreceptor families, it is obvious that a more detailed understanding of the modularity of sensor-effector couples is required. The project Signal Integration in Phytochrome-linked Diguanylyl Cyclases aims at improving the understanding of molecular details involved in red light regulation of diguanylate cyclase activity, which is involved in the formation of a central bacterial signalling molecule. The identification of important signalling elements for the sensory module and regulatory regions of the accompanying effector domain will provide insight into the modular coupling of sensors and effectors frequently observed in nature. We will perform an interdisciplinary approach combining biochemistry with biophysics and structural biology to characterise these systems. Using atomic models obtained by x-ray crystallography and functionally extending their characterization by in solution methods addressing dynamic aspects of the photoreceptor proteins, we will identify functionally relevant elements involved in photo-activation and signal transmission. The combined results will significantly strengthen our understanding of light-signal transduction from the point of the photoreceptors as well as the effector domains. Eventually this will enable a better understanding of the modularity observed in natural light-regulatable systems and support the rational design of artificial optogenetic tools. In the long run, this will allow scientist to probe biological systems with new tools that could enable scientists to address biological questions that are currently not possible.

Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate that plays a central role in bacterial lifestyle decisions. While critical signal integration elements have been described for a set of model system phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. In this project, we showed that dynamics-driven allostery of the dimeric bacteriophytochromes is key to understanding signal integration on a molecular level. We also showed that the overall conformational dynamics of sensor and effector domains are influenced by a multitude of structural elements, to name a few, the light-sensing cofactor environments, accessory elements lining the chromophore environment (PHY-tongue and N-terminal segment), and even the dimer-interface of a domain providing the link to the central coiled-coil sensor-effector linker element. The length of the latter element was shown to be under considerable evolutionary pressure and pronouncedly influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. In addition, we demonstrated the functional importance of mixed functional state dimers using a fast light-to-dark reverting variant that still enables high fold-changes of enzymatic stimulation by red light. This observation supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed-state spectra observed for many phytochrome systems. These insights were obtained by combining biochemical and spectroscopic analyses of several phytochrome activated diguanylate cyclase (PadC) homologs with structural analyses of conformational dynamics using hydrogen-deuterium exchange experiments coupled to mass spectrometry. We observed that the conformational dynamics correlate with the enzymatic activity of these proteins and that the dynamics can be uncoupled from the site of light absorption in specifically designed protein variants. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. Additionally, appreciation of sensor-effector linkers as integrator elements and their coevolution with parts of the sensory modules is a step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.