Periodic Reporting for period 1 - RESPICHAIN (Structure and mechanism of respiratory chain molecular machines)
Período documentado: 2021-09-01 hasta 2023-02-28
Mitochondria are the powerhouses of the cell, synthesizing the energy currency of the cell (ATP) by oxidative phosphorylation, or cellular respiration. Electron transfer through the chain of respiratory enzymes in the inner mitochondrial membrane is coupled to proton translocation across the membrane. The resulting gradient and flow of protons across the membrane drives the unique turbine-like enzyme ATP synthase. Respiratory enzymes are large multi-subunit protein assemblies (complexes I-IV), with complex I being the largest. Their atomic structures are now known, however, even the basic operating principles of some components of the chain, especially complex I, are not yet understood.
NADH:ubiquinone oxidoreductase (CI) is the entry point into the respiratory chains of mitochondria and bacteria. It couples the transfer of two electrons between NADH and quinone to the translocation of four protons across the membrane. The L-shaped CI consists of the hydrophilic matrix arm, where electron transfer takes place, and the membrane arm containing proton translocation channels. This arrangement implies long-range conformational coupling, but the actual (and very intriguing) mechanism is not known. In 2020 we have solved, by cryo-EM, structures of mammalian complex I in several redox states, allowing us to propose the first experiment-based model of the coupling mechanism of complex I. However, many details remained unknown and further exploration of these ideas was required.
Furthermore, in intact mammalian mitochondria the majority of CI is found within respiratory supercomplexes (SCs), and the functional role of these SCs is hotly debated. Previously we have solved structures of respirasome (CICIII2CIV) and CICIII2, but structures (and function) of other supercomplexes remained unknown. Nicotinamide nucleotide transhydrogenase (NNT) works in tandem with the respiratory chain, coupling proton translocation to hydride transfer between NAD(H) and NADP(H), mostly forming NADPH, used to combat reactive oxygen species (ROS). We have determined the first atomic structure of the entire mammalian enzyme and proposed unique mechanism that may involve ~180 degree rotation of a large NADP(H)-binding domain. However, direct experimental evidence supporting it is still lacking.
Therefore, despite the advances in structural knowledge of the respiratory chain, there is still very little understanding of the coupling mechanisms of CI and NNT, as well as of the functional role of SCs and their complete structural organisation. The ambitious overarching aim of this project is to tackle these very important questions, the “grand challenges” of modern biology. It is clear that the mechanisms of both CI and NNT must involve long-range conformational changes, therefore determining (by single particle cryo-EM) the structures of these enzymes captured at different stages of the catalytic cycle will be instrumental in deducing the underlying principles. The conclusions are being verified by site-directed mutagenesis and molecular dynamics (MD) studies. The role of the SCs is studied by resolving their structures in the presence of substrates and inhibitors, to capture different aspects of their interactions, accompanied by functional assays. Cryo-electron tomography on mitochondrial membranes will reveal the organisation of supercomplexes in situ, settling the questions on their putative higher order organisation and its physiological relevance.
As a result of this project, the mechanisms of some of the most intriguing molecular machines in biology should become clear. We will know how exactly electron transfer and proton translocation are so tightly coupled in complex I, despite being completely spatially separated. The molecular organisation, the structure and functional role of various respiratory supercomplexes will become clear. The unique “swivel-to-pump?” mechanism of transhydrogenase will be clarified. Thus, we will achieve a much more complete and rounded understanding of the workings of respiratory chain and mitochondria in general. The results will have far-reaching implications in biology and for society in general, since mitochondrial research is currently coming to the forefront in many medical research areas because of the emerging role of mitochondria not only in energy production but also in biosynthesis, redox homeostasis, oncogenic signaling, innate immunity and apoptosis.
NADH:ubiquinone oxidoreductase (CI) is the entry point into the respiratory chains of mitochondria and bacteria. It couples the transfer of two electrons between NADH and quinone to the translocation of four protons across the membrane. The L-shaped CI consists of the hydrophilic matrix arm, where electron transfer takes place, and the membrane arm containing proton translocation channels. This arrangement implies long-range conformational coupling, but the actual (and very intriguing) mechanism is not known. In 2020 we have solved, by cryo-EM, structures of mammalian complex I in several redox states, allowing us to propose the first experiment-based model of the coupling mechanism of complex I. However, many details remained unknown and further exploration of these ideas was required.
Furthermore, in intact mammalian mitochondria the majority of CI is found within respiratory supercomplexes (SCs), and the functional role of these SCs is hotly debated. Previously we have solved structures of respirasome (CICIII2CIV) and CICIII2, but structures (and function) of other supercomplexes remained unknown. Nicotinamide nucleotide transhydrogenase (NNT) works in tandem with the respiratory chain, coupling proton translocation to hydride transfer between NAD(H) and NADP(H), mostly forming NADPH, used to combat reactive oxygen species (ROS). We have determined the first atomic structure of the entire mammalian enzyme and proposed unique mechanism that may involve ~180 degree rotation of a large NADP(H)-binding domain. However, direct experimental evidence supporting it is still lacking.
Therefore, despite the advances in structural knowledge of the respiratory chain, there is still very little understanding of the coupling mechanisms of CI and NNT, as well as of the functional role of SCs and their complete structural organisation. The ambitious overarching aim of this project is to tackle these very important questions, the “grand challenges” of modern biology. It is clear that the mechanisms of both CI and NNT must involve long-range conformational changes, therefore determining (by single particle cryo-EM) the structures of these enzymes captured at different stages of the catalytic cycle will be instrumental in deducing the underlying principles. The conclusions are being verified by site-directed mutagenesis and molecular dynamics (MD) studies. The role of the SCs is studied by resolving their structures in the presence of substrates and inhibitors, to capture different aspects of their interactions, accompanied by functional assays. Cryo-electron tomography on mitochondrial membranes will reveal the organisation of supercomplexes in situ, settling the questions on their putative higher order organisation and its physiological relevance.
As a result of this project, the mechanisms of some of the most intriguing molecular machines in biology should become clear. We will know how exactly electron transfer and proton translocation are so tightly coupled in complex I, despite being completely spatially separated. The molecular organisation, the structure and functional role of various respiratory supercomplexes will become clear. The unique “swivel-to-pump?” mechanism of transhydrogenase will be clarified. Thus, we will achieve a much more complete and rounded understanding of the workings of respiratory chain and mitochondria in general. The results will have far-reaching implications in biology and for society in general, since mitochondrial research is currently coming to the forefront in many medical research areas because of the emerging role of mitochondria not only in energy production but also in biosynthesis, redox homeostasis, oncogenic signaling, innate immunity and apoptosis.
Overall there was an excellent progress with the project. We have published a paper in Nature (Kravchuk et al. 2022) which outlined for the first time a universal, detailed and experiment-based mechanism of complex I. The mechanism differs dramatically from previous proposals but is very robust and explains all the unusual features of recently accumulated complex I structures. We developed much further our previous proposal (Kampjut and Sazanov, Science 2020) and clarified many questions remaining from that study. Extensive functional and structural (cryo-EM) studies with both bacterial and mammalian sources (Objectives 1 and 2) were performed for this new work. We have solved high-resolution (up to 2.2 Å) structures of Escherichia coli complex I in different redox states including, crucially, catalytic turnover. The proposed novel mechanism comprises a “domino effect” series of proton transfers and electrostatic interactions: the forward wave (“dominoes stacking”) primes the pump and the reverse wave (“dominoes falling”) results in the ejection of all pumped protons from the distal antiporter-like subunit. We believe it represents a seminal milestone in bioenergetics and mitochondrial research and will serve as a basis both for understanding the intricacies of mitochondrial and bacterial metabolism and for future experiments aimed at verifying the details of the mechanism.
Additionally, a detailed characterisation of murine complex I in different conditions (activated, deactivated, re-activated, turnover) is being performed in order to resolve any remaining controversies over the assignment of the so-called “open” state of complex I as either a part of catalytic cycle or off-pathway “deactive” state. We accumulated a lot of data allowing us to definitely assign various structural features specifically to functionally induced “deactive” state. This study is being written up for publication.
Good progress is achieved with Objective 3 on the characterisation of mitochondrial supercomplexes (SC). The structure of SC CIII2CIV was published just at the beginning of the funding period (Vercellino and Sazanov, Nature 2021) and the important review on the organisation of respiratory chain was published already as a part of this project (Vercellino and Sazanov, Nature Rev Mol Cell Biol 2022). Excitingly, we have now identified and determined a structure of a previously unknown type of SC. This study essentially completes the structural characterisation of mammalian supercomplexes repertoire and the manuscript on it is now in the final stages of preparation for submission to high-impact journal.
For Objective 4 on nicotinamide nucleotide transhydrogenase (NNT), many cryo-EM datasets were collected and analysed for T. thermophilus enzyme, and the first high-resolution structure of the entire bacterial enzyme is now being refined and prepared for publication. It confirms that indeed, as predicted, in the bacterial enzyme the two NADP(H)-binding domains are facing in opposite directions, consistent with the mechanism of NNT that we suggested earlier. Other datasets, including NADP(H)-bound and turnover, are being processed. For human NNT the expression of sufficient amounts of protein turned out to be challenging and is still being optimised.
Additionally, a detailed characterisation of murine complex I in different conditions (activated, deactivated, re-activated, turnover) is being performed in order to resolve any remaining controversies over the assignment of the so-called “open” state of complex I as either a part of catalytic cycle or off-pathway “deactive” state. We accumulated a lot of data allowing us to definitely assign various structural features specifically to functionally induced “deactive” state. This study is being written up for publication.
Good progress is achieved with Objective 3 on the characterisation of mitochondrial supercomplexes (SC). The structure of SC CIII2CIV was published just at the beginning of the funding period (Vercellino and Sazanov, Nature 2021) and the important review on the organisation of respiratory chain was published already as a part of this project (Vercellino and Sazanov, Nature Rev Mol Cell Biol 2022). Excitingly, we have now identified and determined a structure of a previously unknown type of SC. This study essentially completes the structural characterisation of mammalian supercomplexes repertoire and the manuscript on it is now in the final stages of preparation for submission to high-impact journal.
For Objective 4 on nicotinamide nucleotide transhydrogenase (NNT), many cryo-EM datasets were collected and analysed for T. thermophilus enzyme, and the first high-resolution structure of the entire bacterial enzyme is now being refined and prepared for publication. It confirms that indeed, as predicted, in the bacterial enzyme the two NADP(H)-binding domains are facing in opposite directions, consistent with the mechanism of NNT that we suggested earlier. Other datasets, including NADP(H)-bound and turnover, are being processed. For human NNT the expression of sufficient amounts of protein turned out to be challenging and is still being optimised.
We consider our recent cryo-EM studies to be beyond the state of art, as we achieved very high, up to 2.2 Å resolution structures of huge membrane protein assemblies of up to 2MDa in a range of different redox conditions, including catalytic turnover, which is missing in most studies from other groups. This allows us to explore the entire conformational landscape of molecular machines, as well as to reliably model all internal water molecules and thus definitively derive potential proton transfer pathways, a key to any mechanistic study of proton pumps.
We expect that in a near future several projects within this programme will result in more high-impact publications, including the structure of a novel respiratory supercomplex, structures of NNT from bacteria and humans, and further detailed mechanistic description of the catalytic mechanisms of complex I and NNT.
We expect that in a near future several projects within this programme will result in more high-impact publications, including the structure of a novel respiratory supercomplex, structures of NNT from bacteria and humans, and further detailed mechanistic description of the catalytic mechanisms of complex I and NNT.