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New magnetic resonance techniques to determine the dynamic structure of mitochondrial outer membrane proteins and their complexes

Final Report Summary - DYNAMOM (New magnetic resonance techniques to determine the dynamic structure of mitochondrial outer membrane proteins and their complexes)

Mitochondria play a key role for a plethora of processes in mammalian cells and many human pathologies are associated with defects in mitochondria such as diabetes, age-related neurodegenerative diseases or cancer. The mitochondrial outer membrane encloses the entire organelle and contains several proteins that are essential for the transport of metabolites and proteins between the cytosol and the mitochondrion. The unique structural and functional characteristics of mitochondria promises to enable the selective targeting of drugs designed to modulate the function of this organelle for therapeutic gain. However, our ability to develop novel, selective and improved therapies for cancer and age-related neurodegeneration is significantly impaired due to a lack of information about the structure of the gatekeeping proteins of the outer mitochondrial membrane. Even less is currently known about the dynamics of membrane proteins despite the fact that dynamics are expected to constitute the essential link between structure and function of membrane proteins.

The major aim of the DYNAMOM proposal was therefore to develop new techniques in NMR spectroscopy and to combine solution NMR with solid-state NMR and other techniques to enrich the sparse information from each of the individual methods and provide insight into the dynamic structure of membrane proteins on a new level.

Many of the methods developed in the DYNAMOM project were novel and unconventional. This includes the method of hydrogen/deuterium exchange coupled to solution-state NMR for the analysis of liposome-embedded membrane proteins, the paramagnetic method of tagging membrane proteins through external reporter proteins and the use of 15N spin relaxation rates to determine the structure and dynamics of mammalian membrane proteins at higher accuracy and resolution.

In addition, the integration of complementary experimental methods was essential, in order to reach the goals of DYNAMOM. This included combination of solution-state NMR with single-molecule force spectroscopy and the combination of solution- and solid-state NMR with molecular dynamics simulations and electrophysiology measurements.

Application of novel methods developed in the DYNAMOM project in combination with state-of-the-art methods in solution- and solid-state NMR spectroscopy to the mitochondrial membrane proteins VDAC, Tom40 and TSPO provided several breakthroughs. This includes the finding that the mechanical flexibility of the VDAC barrel exceeds by up to one order of magnitude that determined for β-strands of other membrane proteins. Through a combination of studies we furthermore revealed a molecular mechanism for the functional regulation of VDAC that is based on changes in the structure and dynamics of the VDAC barrel rather than its N-terminal alpha-helix. A ground-breaking step in the project was also the determination of the three-dimensional structure of the mitochondrial translocator protein TSPO in complex with a ligand, which is used for the diagnosis of neuroinflammation, and the finding that cholesterol-binding modulates the tertiary and quaternary structure of membrane-embedded mammalian TSPO.

Our findings support the important role of solution- and solid-state NMR spectroscopy for the study of conformational changes in small-to-medium sized membrane proteins and highlight the importance of molecular flexibility for the function of mammalian membrane proteins.