Structures, light signaling and allostery mechanisms of photoreceptor kinases - Project Summary Photoreceptor kinases are multi-domain signaling proteins that regulate a wide range of light responses in living organisms. They share similar modular protein architecture with many receptor kinases involved in virulence detection, and cell metabolism, migration, and differentiation. Despite their importance in signal transduction and disease biology, how receptor kinases couple a molecular trigger such as light absorption or ligand binding to a phosphorylation signal remains elusive at both the structural and mechanistic levels. The primary challenge is that these signaling proteins are intrinsically dynamic, largely limiting structural studies to their truncated forms. However, a complete mechanistic understanding requires structural and functional interrogation of full-length proteins in which both sensor and effector domains are present. To address these challenges, we will harness recent advances in cryo-EM single particle analysis to investigate the structures and dynamics of full-length photoreceptor kinases that share key mechanistic aspects with their prokaryotic and eukaryotic counterparts. Our long-term goal is to elucidate the general principles of signaling and allosteric regulation in modular receptor kinases by provoking and resolving functionally relevant structure dynamics. Building on our expertise in photoreceptor research, we will take an integrated approach of structural biology, biochemistry, and spectroscopy to tackle the molecular mechanisms of light-dependent kinase activation in two representative bilin-based photoreceptors - a canonical bacteriophytochrome and a dual-sensor photoreceptor kinase. Our supporting data have revealed light-induced global protein reorganizations in both systems, demonstrating the feasibility of these studies. In this project, we will first determine the full-length structures in different signaling states by resolving structural heterogeneity. We will then establish the functional relevance of the resolved structures by comparing their population change between datasets obtained under different light and ligand conditions. We will also dissect the motions in the central helical spine while addressing the roles of dimer asymmetry and order-disorder transition in long- range signaling via a joint analysis of conformational states captured by cryoEM and crystallography. Last but not the least, we will employ complementary methods of mutagenesis and functional assays to interrogate key interactions underlying the allosteric regulation and signal integration. Findings from the proposed studies will provide unprecedented insights into the molecular events driving the allosteric actions in modular receptor kinases beyond photoreceptors. Bilin-based photoreceptors are photo-switchable, spectrally versatile, and modular in function. They hold great promise in diverse biomedical applications exploiting light to probe cellular processes, modulate biological functions, and treat human diseases. Importantly, the mechanistic understanding gained from these naturally occurring photoreceptors will lay the foundation for ultimately engineering light-activated enzymes of desired signaling logic in novel therapeutic solutions via optogenetics.
