Structural and mechanistic studies of PAS sensing

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ABSTRACT For cells to properly adapt to changing environmental conditions around them, they must rely on sensory proteins which both detect these changes and initiate proper responses. As one solution to this challenge, nature has evolved classes of ligand-regulated protein/protein interaction domains, harnessing these to control numerous types of protein function. Understanding how these domains undertake the requisite sensing and signaling events has given insight into fundamental aspects of biology, enabled the development of new tools for biomedical research, and inspired novel therapies to disease. Here we focus on examining the signaling mechanisms used by proteins containing PAS (Period-ARNT-Singleminded) domains, found in tens of thousands of proteins where they control the activity of over 20 enzymatic and non-enzymatic effector domains. These processes are regulated by different stimuli in different members of the family, harnessing changes in the occupancy or configuration of bound cofactors to “switch” activity on and off. Some aspects of these triggers are well understood in some PAS domains, such as in the subset known as LOV (Light-Oxygen- Voltage) photosensory domains, where blue light illumination drives the specific photochemical formation of protein/flavin adducts which allosterically controls protein conformation around the chromophore. However, fundamental questions regarding these signaling processes remain unanswered, limiting our understanding of how environmental changes are sensed and how these might be artificially controlled. We propose to answer these limitations by pursuing three aims: 1). Determine activation-associated structural changes of several classes of bacterial LOV and PAS proteins; 2). Examine the generality of LOV signaling within a novel class of fungal RGS-LOV proteins; 3). Examine PAS regulation of serine/threonine kinase activity in the nutrient- sensing human PAS kinase. To achieve these ends, we will take advantage of a broad foundation of preliminary structural and functional data that will be extended with a mix of biophysical and biochemical studies. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights into both basic aspects of biomedicine along with potential therapeutic and biotechnology applications.