Atomic-level characterization of self-regulatory mechanisms in large multidomain enzymes

PROJECT SUMMARY/ABSTRACT Enzymes are remarkable nanomachines that play a myriad of essential functions in cellular metabolism. Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of regulation of enzyme function, yet our understanding of the fundamental mechanisms by which concerted protein motion facilitate enzymatic activity is still largely incomplete. Indeed, while several studies have appeared in the past two decades describing how conformational dynamics mediate the biological function of small proteins, our understanding of how the coupling among multiple conformational equilibria determines the activity of large multidomain systems continues to lag. The overall goal of this proposal is using and developing integrated approaches combining NMR with complementary biophysical and biochemical tools to reveal how modulation of local disorder upon cofactor/substrate binding affects concerted motions and regulates the activity of high molecular weight enzymes that are essential for human and bacterial metabolism. This combination of tools sensitive to protein motion brings a newly detailed picture of high molecular weight enzyme function. The enzymes characterized in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the AlkB family of nucleic acid demethylases – together these distinct classes of enzymes show how the relationship between local disorder and concerted domain motions can be probed by this combination of tools and demonstrate how essential these mechanisms are across diverse enzyme classes. In particular, the EI enzymatic activity depends upon the synergistic action of four conformational equilibria that results in a series of large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding. Therefore, our efforts to uncover EI function at atomic level will reveal how modulation of local disorder mediates long-range interdomain communication and, ultimately, regulates the activity of this essential bacterial enzyme. The AlkB dioxygenases are flexible enzymes that are known to undergo modulation of their internal dynamics upon substrate binding. Our studies will visualize conformational disorder in apo and holo AlkB enzymes with unprecedented atomic-resolution details, and will reveal how residual disorder at the active site determines substrate selectivity. In addition, we will investigate a number of complexes formed by AlkB proteins with their inhibitors. We expect these results to indicate new strategies, based on selective perturbation of conformational disorder, to develop AlkB inhibitors with subfamily selectivity. In summary, my research program will elucidate the coupling between large scale conformational changes and function in two distinct classes of high molecular weight multidomain enzymes, providing new insights for future therapies for obesity and cancer as well as novel antibiotic targets. Moreover, these efforts pushing the frontier of the application of biophysical tools to study with atomic resolution the relationship between disorder and functional concerted motions in complex enzymes provide a template transferrable to mechanistic investigations of uncharted multi-domain systems.