Structural and dynamic insights into the mechanisms of mitochondrial AAA proteases

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Mitochondria are the energy powerhouses of the eukaryotic cells and play prominent roles in a number of essential metabolic and signaling pathways. Due to the intense metabolic activities occurring inside mitochondria, an efficient and targeted protein quality control system in mitochondria is vital for the functional integrity of the organelle and critical for overall cell survival. Imbalances in the mitochondrial proteostasis have been implicated in neurological diseases, cardiovascular disorders, and cancer. Central to mitochondrial protein quality control is a network of mitochondrial proteases which degrade undesired and damaged proteins in a regulated fashion. The long-term objective of my research is to understand how various mitochondrial proteases function from a structural and dynamic perspective and to uncover how they work in a specialized and concerted manner to maintain mitochondrial proteostasis under normal and stressed conditions.     In the proposed research, we will focus on two conserved ATP-dependent mitochondrial proteases, m-AAA and i-AAA, referred to as mitochondrial AAA proteases, which target substrates in the mitochondrial intermembrane, the matrix and the intermembrane space, and play essential roles in maintaining mitochondrial proteostasis. Defects in these proteases have been implicated in neurological and neurodegenerative disorders as well as metabolic dysfunctions. The proposal describes a thorough structural and dynamical characterization of the mitochondrial AAA proteases using solution nuclear magnetic resonance (NMR) spectroscopy as the main approach in conjunction with other biophysical and biochemical techniques, designed to gain mechanistic insights into how the proteases carry out and regulate their functions. To achieve this goal, we propose the following specific aims: (1) identify interaction sites between the AAA proteases and their substrates, and determine the mechanism of substrate recognition; (2) identify and characterize functionally important structural features and conformational dynamics; (3) determine allosteric pathways connecting structural motifs and characterize domain communications; (4) understand cooperativity between different subunits from a structural perspective and its roles in ATP hydrolysis and unfolding/proteolytic functions. This proposed research program will provide a fundamental understanding of how the AAA proteases function and how disease mutations may lead to impairment of their enzymatic activities, and ultimately shed light into how protein quality control is achieved in mitochondria. Extensive training opportunities will be provided for HQPs not only to acquire expertise in protein NMR spectroscopy but also to be proficient in structural and functional characterization of proteins using a variety of other biochemical and biophysical tools. We hope to nurture a future generation of scientists to flourish in both academic and industrial careers.