Deciphering the mechanisms underlying the operational plasticity of AAA+ motors

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AAA+ (ATPases associated with various cellular activities) motors are powerful biomolecular machines found in all kingdoms of life that couple ATP binding and hydrolysis to drive conformational changes for the mechanical translocation of a variety of cellular substrates. They are critical in many cellular processes including protein degradation, protein disaggregation, and DNA replication. As such, there is significant interest in understanding the molecular basis of AAA+ motor function. Despite numerous high-resolution structures over the past decade, this has proven to be a formidable challenge. Based on these structures, a `hand-over-hand' model has been proposed where sequential ATP hydrolysis in hexameric AAA+ motors results in movement of bulky pore loops along the substrate and unidirectional substrate translocation. By contrast, the probabilistic model posits that individual subunits hydrolyze ATP asynchronously, and the sequence of ATP hydrolysis is not restricted to a defined order. These competing models illustrate the limits of inferring biochemical mechanisms of highly dynamic systems, such as AAA+ motors, from static protein structures and demonstrate the need for the application of complementary methodologies capable of probing structural plasticity. The long-term goals of my research program are to understand how the structure and dynamics of all seven evolutionary clades of AAA+ motors are linked to their function, and to uncover and characterize their interactions with regulatory and substrate molecules. Here I focus on representative members of the HCLR clade (HslUV, ClpAB-D2, Lon, and RuvB family), the highly conserved Lon and HslUV AAA+ proteases, for which high-resolution structures are available yet many outstanding questions remain. This proposal describes a comprehensive structural and dynamical characterization of the Lon and HslUV proteases using a combination of solution on nuclear magnetic resonance (NMR) spectroscopy and hydrogen deuterium exchange mass spectrometry (HDX-MS). These two methods, together with biochemical techniques, provide unique mechanistic insights into the function of AAA+ motors. I propose the following short- and medium-term specific aims: (1) Understanding the role of cooperativity in AAA+ motors; (2) Characterization of substrate binding to Lon and HslUV; and (3) Characterization of structural dynamics and functional allostery in AAA+ proteins. Insights into protein dynamics obtained via my combined NMR-MS approach complement existing X-ray and cryo-EM structures and promise to provide a detailed view of the function of AAA+ motors and mechanochemical enzymes in cells. These three carefully integrated aims, combined with my HQP Training Plan that focuses on flexible mentorship to accommodate different HQP learning styles, needs, and backgrounds, guarantees a fertile ground for training the next generation of scientists.