Light Regulated Two-Component Signal Transduction

Prokaryotic intracellular signaling commonly relies on two-component signal transduction, involving pathways of phosphorylation between histidine kinases (HK) and response regulators (RR). Environmental stimuli activate the HK, which undergoes an autophosphorylation reaction and subsequent phosphotransfer to downstream RRs that change cellular physiology. Here we report our studies of HKs linked to Light-Oxygen-Voltage (LOV) domains, a class of blue light photosensors, and the RRs they target. One such HK is found within the marine bacteria Erythrobacter litoralis, EL362 contains a non-conservative natural G150R point mutation in the center of the LOV domain. We hypothesize that this prevents EL362 from binding the flavin chromophore that is necessary for photosensing. R1 is a single domain RR located adjacent to EL362 in the E. litoralis genome. All proteins were overexpressed in E. coli, purified using Ni⁺² affinity and gel filtration chromatography. Solution NMR spectroscopy was used to monitor conformational changes in EL362 and R1. Multiangle Laser Light Scattering was used to determine the molecular weight and oligomeric state of the proteins. Autophosphorylation and phosphotransfer assays were performed on the HK and RR respectively to measure activity. Our results show that EL362 R150G undergoes canonical LOV photochemistry, while R150 is unable to bind FMN and undergo the dark/light cycle expected of proteins with this type of sensor. Upon phosphorylation by EL362, R1 undergoes a significant conformational change but remains monomeric. R150G mutation is sufficient to restore light regulation and increase kinase activity. EL362 targets R1 for phosphorylation, which causes R1 to undergo dramatic conformational changes.