Structural studies of disorder-based protein-protein interactions

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Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not adopt a well-folded structure but yet retain function. Therefore, they challenge the classical protein structure-function paradigm. Importantly, IDPs are highly abundant in all organisms and play crucial roles in many important biological processes. The long-term goal of my research program is to understand the complex structure--function relationship of IDPs. I am particularly interested in IDPs that are involved in cell signalling pathways, in which they often act as hubs in protein-protein interaction networks. My group aims to unravel the structural and dynamical characteristics of IDPs, and the ways they interact with different binding partners at the molecular level. The knowledge is important for understanding how IDPs function, and the cellular processes they regulate. In the next 5-year grant cycle, my research will be focused on 1) the mechanisms by which IDPs function as protein hubs in recruiting binding partners, and 2) the effects of post-translational modifications, especially phosphorylation, on the structural and target recognition properties of IDPs. Our studies will particularly focus on Nrf2. Nrf2 is the key transcription factor that orchestrates the cellular responses to oxidative stress. Its activity is tightly regulated via a cascade of protein-protein interactions. Many of these interactions are modulated by phosphorylation, adding an additional level of complexity to the Nrf2 regulation. We will combine experimental and computational approaches in dissecting the mechanisms by which Nrf2 interacts with several targets that play critical roles in Nrf2 regulation. Techniques including nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, genetic code expansion, and molecular dynamic (MD) simulation will be used. NMR spectroscopy is the most powerful biophysical technique for studying IDPs at the molecular level. With the advancements in computational power and simulation methodologies, MD simulation is quickly emerging as an excellent complementary approach for obtaining molecular insights of IDP. We will also investigate the effects of phosphorylation on the structure and target recognition of Nrf2. Despite the continued advances in structural biology, molecular understanding about the impact of phosphorylation on protein conformation and target binding is impeded by the inability to synthesize site-specifically phosphorylated proteins with high homogeneity and in large quantity. To overcome this technological barrier, we will use enzymatic phosphorylation and an innovative genetic code expansion method to site-specifically install phosphoserine at the desired locations in Nrf2. The studies will establish structural basis of phosphorylation as functional switches of Nrf2.  Together, results of the proposed research will make a significant contribution to the exciting IDP field.