Characterization of Sequence-Structure-Function Relationships in Phytochrome Regulated Diguanylate Cyclases

The three-dimensional structure of proteins influences function in many ways, such as substrate binding site architecture or accessibility of cofactor binding pockets defining enzyme functionality. Additionally, protein dynamics frequently play an important role in signal transduction processes of sensor-effector systems. In a subfamily of bacterial photoreceptors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic linker element connects sensory to output modules and plays an essential role in signal integration. We show that length and composition of this linker strongly influence sensor-effector interaction and as such are under considerable evolutionary pressure. Most importantly, the linker length, in interplay with the upstream PHY-specific domain, influences the efficiency of effector activation and can even cause light-induced enzyme inhibition. Compatibility of the sensory core and the linker element is essential for efficient light-controlled regulation of enzymatic activity. We further demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. Characterization of PadC homologs with different linker architectures revealed that biochemical behaviour is similar but non-homogenous for all phylogenetic branches excluding a small sub-family of PadCs characterised by a linker length breaking the heptad pattern. Differences in light-induced regulation of enzymatic function as well as spectral behaviour correlate with this unusual linker architecture. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with the sensory module is a further step towards use of functionally diverse homologs as framework for rationally designed optogenetic tools.