The AppA protein in Rhodobacter sphaeroides is an antagonist of the transcriptional repressor protein PpsR, which can block the transcription of several photosynthesis genes through binding to a consensus sequence. To date, the AppA protein is unique in its ability to sense and transmit both light and redox signals.
The AppA protein consists of an N-terminal FAD-binding domain, recently named BLUF (sensors of blue light using FAD), and a C-terminal domain, which is involved in the light- and redox-dependent regulation of photosynthesis genes. The BLUF domain is a new family of blue light photoreceptors and it is present either in multidomain proteins or as a short protein only consisting of the BLUF domain. Recently, four BLUF domains have been found in the photoreceptor protein photoactivated adenylyl cyclase (PAC) from the eukaryotic organism Euglena gracilis. Each BLUF domain is linked to a downstream domain whose function is completely different from that of the C-terminal domain of AppA.
In this work, a hybrid protein containing one of the BLUF domains from the PAC protein and the C-terminal domain of AppA was expressed in R. sphaeroides and the function of this hybrid protein was investigated. The results showed that the BLUF domain from the PAC protein can fully replace the AppA-BLUF domain in the light-dependent signaling. Moreover, this work revealed that the signal transmission by AppA does not require covalent linkage of the BLUF domain and the C-terminal domain. These two observations demonstrate that the BLUF domain is fully modular and can relay signals to completely different output domains. It was previously assumed that the small BLUF-containing proteins transfer the light-dependent signals to other proteins by protein-protein interactions. The interaction between the BLUF domain and the C-terminal domain of AppA revealed in this work supports this assumption.
The previous studies have shown that AppA interacts with PpsR and inhibits the repressor activity of PpsR. However, the interacting domains of AppA and PpsR have not been determined. It was found in this study that the C-terminal domain of AppA is sufficient to inhibit the DNA-binding activity of PpsR in vivo and in vitro. This observation indicates that the binding site between AppA and PpsR must be located at the C-terminal domain of AppA. Furthermore, surface plasmon resonance (SPR) based interaction studies demonstrate that the C-terminal domain of AppA can interact with PpsR in the absence of the BLUF domain. In addition, this work presented the first evidence that the C-terminal domain of AppA contains a heme as the redox sensitive prosthetic group. Mutant analysis revealed that some of the amino acids in the heme-binding domain are essential for the function of AppA. It was also found that the cofactor heme affects not only the interaction of the C-terminal domain of AppA with PpsR but also the interaction of the C-terminal domain of AppA with the BLUF domain. These data demonstrate that the cofactor heme plays a key role in the light- and redox-dependent signaling.
Besides the AppA/PpsR system, several other regulatory systems in R. sphaeroides also control the expression of photosynthesis genes according to light and redox conditions. Thus, the interplay between the AppA/PpsR system and other systems was investigated in this work. The results showed that i) the AppA/PpsR system is the major contributor that mediates the expression of photosynthesis genes in response to light, ii) the PrrB/PrrA system is sufficient to activate photosynthesis gene expression in response to the decrease of oxygen and the redox-dependent regulation by the AppA/PpsR system requires the presence of the PrrB/PrrA system, and iii) the deficiency of thioredoxin causes the increase in the redox potential and consequently affects the AppA-PpsR system in light-dependent signaling.
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