Structural basis of signal transduction within PAS regulated Ser/Thr kinase.

Abstract

To control integral cellular processes, nature has developed molecular switches where the sensory domain senses external cues and controls the function of relevant effector domains. Here, we focus on the superfamily of PAS (Per-ARNT-Sim) sensory domains and their roles in regulating Ser/Thr Kinases (STKs). PAS domains achieve regulatory control by internally binding ligands/cofactors (e.g., O2 binding to heme in FixL; flavins within the Light-Oxygen-Voltage (LOV)-type PAS domains). While aspects of PAS sensing are understood, the signaling mechanism from sensory to effector domain and Protein-Protein Interactions (PPIs) within full-length proteins or complexes remain elusive. To address these, we investigated plant phototropin, Avena sativa Phototropin1 (AsPhot1). AsPhot1 has two LOV domains followed by the STK domain, where the LOV2 regulates the kinase function to maintain essential plant physiology via blue light sensing. We used biochemical assays in combination with HDX-MS to study the signal transduction from LOV2 to the STK domain. Our data shows substantial conformation changes in the dark vs. light state upon photoactivation, highlighting key regions might be necessary for LOV-regulated kinase functions. We also observed the activation of kinase involves destabilizing the linker region between LOV2-STK, which interacts with the n-lobe of the kinase near the ATP binding site. We speculated that upon illumination, rearrangement of these linkers might hold the key to the STK substrate association and downstream functions. Additionally, using a low-resolution cryo-EM map, combined with local HDX-MS data and computational resources (e.g., AlphaFold), we built a model for the LOV2-STK complex. Our model highlights critical PPI sites within AsPhot1 and provides additional insights into understanding the generality of how protein kinases interact with their associated domain via conserved residues and subdomains. These findings may help develop novel optogenetic tools and therapeutics for mammalian PAS regulated kinase.