Periodic Reporting for period 1 - MitoMemInsert (Structural determination and dynamics of the mitochondrial import protein (MIM) by cryo-EM and magic-angle-spinning (MAS) nuclear magnetic resonance (NMR))
Reporting period: 2022-10-01 to 2024-09-30
Mitochondria are involved in many cellular processes, including numerous essential metabolic reactions, sensing, ageing and cell death. They are particularly well known as the "powerhouse" of the cell, because they generate the energy, in form of adenosine triphosphate (ATP) which is used as the main "energy currency" of the cell. Mitochondria are believed to stem from a merging event, billions of years ago, of a bacterium (now the mitochondrion) with a predecessor of archaeal cell, which is thought to be the event that allowed for eukaryotic life, and thus all higher forms of life, to emerge. Along evolution, mitochondria have transferred almost the entirety of they genetic material to the nucleus, where it is better protected from damage than in the vicinity of the energy-producing machineries of the mitochondria. Consequently, 99% of the proteins that work in mitochondria are encoded in the DNA in the nucleus, and are made outside the mitochondrion, in the cytoplasm. An important question in this context is therefore how all these proteins are imported into the mitochondrion, i.e. brought from their place of synthesis in the cytosol to their final "work place" in the mitochondrion. This import is particularly tricky for mitochondrial membrane proteins: they are inherently insoluble in water and therefore they need a tightly controlled "shuttle" to avoid them from aggregating in the cytoplasm. Moreover, they need a machinery which inserts them into their final destination in the membrane (outer or inner mitochondrial membrane), and possibly also a channel to cross the outer membrane of the mitochondrion, namely for those membrane proteins that ultimately get inserted into the inner membrane. As the proteins are initially unfolded, namely right after their synthesis, they also need to refold to the correct three-dimensional structure in their final destination.
Cells have developed a complex machinery to safely import, refold and insert mitochondrial proteins. This is essential for eukaryotic life, because failure of import of proteins can have catastrophic consequences for the cell; dysfunctional mutations in the import-machinery components is often lethal very early on, and even small deficiencies of import are related to human diseases. Deciphering the function of the machineries involved in mitochondrial import is, therefore, not only of fundamental interest but possibly has implications for understanding human diseases.
The goal of this project was/is to characterize a so-called membrane-protein insertase of mitochondria, in terms of its 3D structure, function and interaction with to-be-inserted membrane proteins.
Cells have developed a complex machinery to safely import, refold and insert mitochondrial proteins. This is essential for eukaryotic life, because failure of import of proteins can have catastrophic consequences for the cell; dysfunctional mutations in the import-machinery components is often lethal very early on, and even small deficiencies of import are related to human diseases. Deciphering the function of the machineries involved in mitochondrial import is, therefore, not only of fundamental interest but possibly has implications for understanding human diseases.
The goal of this project was/is to characterize a so-called membrane-protein insertase of mitochondria, in terms of its 3D structure, function and interaction with to-be-inserted membrane proteins.
A key steps of this work was to develop the production of the membrane-protein insertase for its study by structural-biology techniques. A further key step was to collect data with electron microscopy, crystallography and/or NMR which provide insights into its 3D structure, interactions and dynamics.
In this project, the first step has been successfully achieved, and the second step has been partly achieved, and is currently in progress. We have used a recombinant protein production scheme to obtain the protein in milligram quantities, and tested successfully different means of reconstituting the protein in a membrane-mimicking environment (such as detergent micelles) and in lipid bilayers (liposomes, nanodiscs). Extensive tests of stability and proper folding of the protein have been achieved. The protein, purified to high chemical purity and conformational homogeneity, has been obtained in several such environments, and we could obtain samples ready for structural analysis.
Electron microscopy has been performed. The analyses of the cryo-EM data, as well as NMR data, has been started and while not yet at a level of publication, we have achieved several tangible outcomes which will serve as the basis for a future publication:
• Cryo-EM data of the protein in complex with a nanobody have provided a first low-resolution insight into the protein
• NMR spectra of the membrane protein show high resolution and open the way to probing at atomic resolution the dynamics and interactions of the membrane-protein insertase.
• The production of the membrane protein has been established which will also form the basis of a technical publication.
In this project, the first step has been successfully achieved, and the second step has been partly achieved, and is currently in progress. We have used a recombinant protein production scheme to obtain the protein in milligram quantities, and tested successfully different means of reconstituting the protein in a membrane-mimicking environment (such as detergent micelles) and in lipid bilayers (liposomes, nanodiscs). Extensive tests of stability and proper folding of the protein have been achieved. The protein, purified to high chemical purity and conformational homogeneity, has been obtained in several such environments, and we could obtain samples ready for structural analysis.
Electron microscopy has been performed. The analyses of the cryo-EM data, as well as NMR data, has been started and while not yet at a level of publication, we have achieved several tangible outcomes which will serve as the basis for a future publication:
• Cryo-EM data of the protein in complex with a nanobody have provided a first low-resolution insight into the protein
• NMR spectra of the membrane protein show high resolution and open the way to probing at atomic resolution the dynamics and interactions of the membrane-protein insertase.
• The production of the membrane protein has been established which will also form the basis of a technical publication.
We have achieved an important step of this project, namely the production of high-quality samples which will eventually allow achieving all the goals presented in the project proposal. While more time is needed, beyond the project duration, in order to get the high-resolution structure, the results obtained here are a solid foundation for deciphering the mechanisms of this membrane protein.