Structural Basis of Thermogenic Mitochondrial Respiratory Complexes Assembly

Project Summary Obesity is the major leading risk factor for type 2 diabetes (T2D) that constitutes a major pandemic. Obesity and T2D are major risks for cardiovascular diseases, viral infections, or cancer. Management of obesity includes exercise, dietary regimens, bariatric surgery, and prescribed medications such as orlistat or GLP-1 agonists. However, not all different strategies are possible, and success is limited because of genetics, motivation, or excessive cost. Body weight is defined by an energy balance equation composed by the food-derived energy minus energy expenditure that includes physical and thermogenic activities. Positive imbalances in this equation cause overweight and obesity but increases in energy expenditure reduces weight and obesity. Thermogenic activity occurs in response to cold temperatures or excess of high calorie diets in specialized cells, brown and beige adipocytes. Beige-like adipocytes, similar to murine beige fat, are present in humans and are activated in response to lower temperatures or beta-adrenergic agonists. Thermogenic function in these specialized cells depends on mitochondrial respiration that produces sufficient proton pumping and gradients dissipated through uncoupling (UCP1) or used to synthesize ATP used in futile reactions that generate energy as heat. Mitochondrial respiration is executed through complexes that form the electron transfer chain ending at molecular oxygen and water. These respiratory complexes (I, III and IV) are present in higher order assemblies forming super- and megacomplexes, however the regulatory and functional consequences of these assemblies is unknown. In preliminary studies, we used unprecedented cryo-EM and computational approaches to identify a novel brown fat cold-inducible respiratory complex super-assembly that represents a new structure of complex I and complex III dimers (Type 2 complex). This new Type 2 complex is absent in brown fat mitochondria in thermoneutral conditions or cold exposed PERK deficient mice that are unable to survive cold temperatures. Based on cryo- EM structural analysis and Quantum Mechanics and Molecular Dynamic simulations, rotation in Type 2 complex forms a highly active class with efficient CoQ electron transfer consistent with cold temperature thermogenic demands. The premise of this application is that cold temperatures or high calorie diets promote the formation of highly active respiratory complexes super-assemblies that support the thermogenic adipocyte function to protect body temperature and obesity/T2D. We propose three aims: 1) determine the structure of brown fat mitochondria respiratory megacomplexes during adaptation to cold temperatures and high calorie diets; 2) structural analysis of respiratory complexes in beige fat during adaptation to cold temperatures or β3-agonist treatment; 3) mitochondrial and respiratory megacomplexes lipid analysis during adaptation to cold temperatures and high calorie diets. The outcomes will provide unprecedent structures of respiratory complexes in highly active thermogenic conditions driven by lower temperatures or excess calorie intakes. Understanding the molecular basis of these structural mechanisms have important implications for obesity and T2D.