title: Engineering and Characterization of RNA-binding LOV photoreceptors
Creators: name: Ziegler, Thea
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: sensory photoreceptors
subject: signal transduction
subject: protein engineering
subject: Light-Oxygen-Voltage (LOV) domains
subject: RNA
subject: RNA-binding proteins
subject: x-ray crystallography
subject: 540

description: Light-Oxygen-Voltage (LOV) photoreceptors are light-sensitive signaling proteins that provide responses to light in the ultraviolet and blue regions of the spectrum. The light stimulus is transmitted to the functional output via structural rearrangements within the light-sensing domain, which consequently modulates the activity of the signaling protein. In recent years, the compact LOV modules have become popular scaffolds for constructing new optogenetic tools. The latter enable precise spatiotemporal control over diverse biological targets in a light-dependent manner. While many photoreceptors convey control over DNA-associated processes, so far there is no candidate that directly acts on RNA. The aim of this work was therefore to provide a novel approach for the light-controlled regulation of RNA molecules, either by identifying and characterizing a naturally occurring photoreceptor that fulfils the desired property of light-regulated RNA binding, or by recombining a well-characterized LOV photosensor with a suitable RNA-binding output domain. Searching for previously uncharacterized photoreceptor candidates in the sequence databases, we discovered a promising gene entry in the gram-positive actinobacterium Nakamurella multipartita. The putative protein product comprises an N-terminal PAS (Per-ARNT-Sim) domain, followed by an RNA-binding ANTAR (AmiR and NasR transcription antitermination regulator) domain and a Cterminal LOV domain, accordingly referred to as 'PAL'. Based on the domain arrangement, we assumed that the RNA-binding function of the ANTAR domain may be controlled by the blue-lightresponsive LOV domain. We thus amplified the PAL gene from the genomic DNA of N. multipartita and confirmed its sequence identity via DNA sequencing. Next, PAL was heterologously expressed in Escherichia coli and purified via immobilized ion affinity chromatography. The purified PAL contains a flavin chromophore and undergoes the characteristic LOV photochemistry after blue light activation. We then applied SELEX (Systematic Evolution of Ligands by EXponential enrichment) to determine specific RNA target sequences for PAL and found two different motif families defined by a common consensus sequence. The five most promising variants were analyzed with the help of electrophoretic mobility shift assays for their binding properties to PAL. The PAL photoreceptor demonstrates a blue-light-triggered binding activity for all of the thereby tested constructs with an apparent KD of around 0.25 µM for the best-binding aptamer under light conditions, which represents an approximate 30-fold increase compared to the corresponding binding activity in the dark. By optimizing the best-binding aptamer, we achieved binding affinities in the nanomolar range (30 ± 3 nM). Regarding the structural and mechanistic investigations of the PAL photoreceptor, we succeeded in obtaining the full-length crystal structure of PAL in its dark-adapted state with a resolution of 2.75 Å. The three-dimensional structure illustrates how signal transmission can be achieved within a LOV photoreceptor with the unusual domain topology of an N-terminally positioned output domain. With a combination of SEC-MALS (Size Exclusion Chromatography 10 combined with Multi-Angle-Light-Scattering) and SEC (Size Exclusion Chromatography) experiments, we demonstrated that both the full-length PAL protein, as well as the isolated LOV domain, occur as a dimer in solution independently of the light conditions. Moreover, we used the architecture of PAL as a design template for the development of further light-regulated RNA-binding proteins with an altered sequence specificity, and were able to generate light-sensitive constructs by replacing the PAL ANTAR domain with that of AmiR from Pseudomonas aeruginosa. The results indicate a great potential of PAL for use in optogenetic applications, as it opens the possibility of generating lightdependent RNA-protein interactions with high affinity. In addition, the structural studies on PAL provide valuable mechanistic insights that will facilitate the improvement of PAL as an optogenetic tool, as well as the construction of novel PAL-oriented chimeric photoreceptor variants.