Periodic Reporting for period 5 - Chap4Resp (Catching in action a novel bacterial chaperone for respiratory complexes)
Periodo di rendicontazione: 2021-10-01 al 2022-03-31
Respiration is the principal way a cell produces energy to fuel its activity. In this process, electrons released from nutrients, transit through a molecular wire of metal clusters and cofactors embedded in respiratory complexes in the inner membrane of mitochondria and bacteria, to an end acceptor. The proton transfer across the membrane, accompanying the electron flow, creates a gradient that provides the energy required to generate ATP and thus to power essential processes of life. Deficiency in integrity and activity of bacterial respiratory complexes makes the bacteria resistant to an important class of antibiotics, aminoglycosides, which require proton gradient for uptake; problems with mitochondrial resporatory complexes cause ageing and diseases. Therefore, understanding the molecular mechanisms of the respiratory complexes biogenesis is an important goal, particularly challenging because of the membrane location of these enzymes, their intricate multisubunit composition and cofactor insertion.
The first and the largest enzyme of the respiratory chain is Complex I (CI), which core is highly conserved from bacteria to humans. This project focused on the structure-function relationships of a three-component protein system proposed to be involved in CI assembly is enterobacteria. In parallel, as a part of a larger collaborative effort, the structure and function of the mitochondrial CI assembly (MCIA) complex was addressed. The project overturned the previous view on the bacterial LdcI-RavA-ViaA triad and resulted in novel models of its mechanism of action, setting the stage for future investigations of an unprecedented molecular network that links the triad with bacterial stress adaptation, membrane homeostasis, respiratory complexes and aminoglycoside bactericidal activity. In addition, we provided a structural basis for the MCIA complex architecture and suggested a novel mechanism for coordinating the regulation of the mitochondrial energetic pathways, with implications on the molecular etiology of the Alzheimer’s disease.
The first and the largest enzyme of the respiratory chain is Complex I (CI), which core is highly conserved from bacteria to humans. This project focused on the structure-function relationships of a three-component protein system proposed to be involved in CI assembly is enterobacteria. In parallel, as a part of a larger collaborative effort, the structure and function of the mitochondrial CI assembly (MCIA) complex was addressed. The project overturned the previous view on the bacterial LdcI-RavA-ViaA triad and resulted in novel models of its mechanism of action, setting the stage for future investigations of an unprecedented molecular network that links the triad with bacterial stress adaptation, membrane homeostasis, respiratory complexes and aminoglycoside bactericidal activity. In addition, we provided a structural basis for the MCIA complex architecture and suggested a novel mechanism for coordinating the regulation of the mitochondrial energetic pathways, with implications on the molecular etiology of the Alzheimer’s disease.
The LdcI-RavA-ViaA triad was synergistically analysed using complementary biophysical, biochemical, structural, fluorescence imaging, and bioinformatics approaches. Structures of decameric Ldcs from different pathogenic and commensal bacteria were solved and compared, functional characterisation of these enzymes performed and a phylogenetic analysis of the parent superfamily accomplished. The acid stress-induced polymerisation of the E. coli LdcI and the conservation of the molecular determinants of LdcI polymerisation in enterobacteria were revealed, and the investigation of the role of the supramolecular assembly in the superfamily undertaken. The structures of the E. coli RavA ATPase in two different states were solved, and the inferred mechanism of ATP hydrolysis extended to the entire family and its corresponding AAA+ ATPases’ clade. The structure of the unique LdcI-RavA cage-like complex was determined, and the ViaA protein and the RavA-ViaA interaction characterised. However, no direct interaction between RavA/ViaA and respiratory complexes could be detected. Excitingly however, a direct interaction of RavA and ViaA with specific inner membrane lipids was discovered and analysed both in vitro and in vivo. The lipid-binding sites were identified, and the effects of RavA/ViaA on cellular lipid homeostasis and membrane morphology addressed. Moreover, RavA/ViaA lipid-binding propensity was directly linked to their effect on the bactericidal activity of aminoglycosides (AGs) under anaerobiosis.
Our findings led us to propose that RavA and ViaA chaperone certain respiratory complexes indirectly, by acting on lipid microdomains in which these complexes are inserted. This hypothesis aligns with our observations on the in cellulo distribution of LdcI. In addition to opening exciting research directions on the links between the LdcI-RavA-ViaA triad and bacterial stress adaptation, respiration, membrane homeostasis and aminoglycoside bactericidal activity, these results improve our knowledge of enterobacterial pathways mobilised in response to AGs under anaerobiosis. Considering that AG efficiency is dramatically reduced in anaerobic conditions encountered by enteric pathogens inside their human host, elucidation of mechanisms allowing for the usage of decreased dosage and consequently lesser toxicity may lead to safer use of this family of antibiotics against a wider range of infections.
Because the direct interaction between the LdcI-RavA-ViaA triad and respiratory complexes could not be confirmed, we decided to extend our work to the MCIA complex, proposed to be functionally analogous to the LdcI-RavA-ViaA triad but undoubtedly binding mitochondrial Complex I. We solved the structures of one isolated protein of the MCIA complex and of its binary subcomplex with another MCIA partner, and revealed a novel mechanism of regulation, crucial for efficient energy production in mitochondria. This set us on track for elucidating the role played by the MCIA complex in CI assembly with a goal to shed light on the mitochondrial bioenergetic pathways and their role in physiology and pathology, particularly in Alzheimer’s disease.
Finally, the project contained sections on methodological development for cryo-ET that we initially planned to use for identification of the LdcI-RavA cage inside E. coli minicells. While we successfully designed and characterised a minicell-producing strain suitable for cryo-ET, the LdcI-RavA complex partitioned uniquely in the mother cells. Thus, instead of the LdcI-RavA complex, we benchmarked the minicells and our cryo-ET image analysis tools that offer streamlined interaction between state-of-the-art software packages, by solving the structure of the core signalling unit of the E. coli chemosensory array. In addition, we showed that our cryo-ET image analysis framework can successfully result in an atomic resolution structure solved from a publicly available dataset ( EMPIAR-10164) commonly used for benchmarking. We created a comprehensive step-by-step guide to obtaining this structure and offered it on a collaborative, online ressource https://teamtomo.org/ that we established as a platform for sharing knowledge about cryo-ET data processing. This platform is now widely used and other researchers contribute their expertise for the common benefit of the growing cryo-ET community.
Our findings led us to propose that RavA and ViaA chaperone certain respiratory complexes indirectly, by acting on lipid microdomains in which these complexes are inserted. This hypothesis aligns with our observations on the in cellulo distribution of LdcI. In addition to opening exciting research directions on the links between the LdcI-RavA-ViaA triad and bacterial stress adaptation, respiration, membrane homeostasis and aminoglycoside bactericidal activity, these results improve our knowledge of enterobacterial pathways mobilised in response to AGs under anaerobiosis. Considering that AG efficiency is dramatically reduced in anaerobic conditions encountered by enteric pathogens inside their human host, elucidation of mechanisms allowing for the usage of decreased dosage and consequently lesser toxicity may lead to safer use of this family of antibiotics against a wider range of infections.
Because the direct interaction between the LdcI-RavA-ViaA triad and respiratory complexes could not be confirmed, we decided to extend our work to the MCIA complex, proposed to be functionally analogous to the LdcI-RavA-ViaA triad but undoubtedly binding mitochondrial Complex I. We solved the structures of one isolated protein of the MCIA complex and of its binary subcomplex with another MCIA partner, and revealed a novel mechanism of regulation, crucial for efficient energy production in mitochondria. This set us on track for elucidating the role played by the MCIA complex in CI assembly with a goal to shed light on the mitochondrial bioenergetic pathways and their role in physiology and pathology, particularly in Alzheimer’s disease.
Finally, the project contained sections on methodological development for cryo-ET that we initially planned to use for identification of the LdcI-RavA cage inside E. coli minicells. While we successfully designed and characterised a minicell-producing strain suitable for cryo-ET, the LdcI-RavA complex partitioned uniquely in the mother cells. Thus, instead of the LdcI-RavA complex, we benchmarked the minicells and our cryo-ET image analysis tools that offer streamlined interaction between state-of-the-art software packages, by solving the structure of the core signalling unit of the E. coli chemosensory array. In addition, we showed that our cryo-ET image analysis framework can successfully result in an atomic resolution structure solved from a publicly available dataset ( EMPIAR-10164) commonly used for benchmarking. We created a comprehensive step-by-step guide to obtaining this structure and offered it on a collaborative, online ressource https://teamtomo.org/ that we established as a platform for sharing knowledge about cryo-ET data processing. This platform is now widely used and other researchers contribute their expertise for the common benefit of the growing cryo-ET community.
Progress beyond the state of the art has been achieved in numerous tasks of the project. From the structural biology point of view, many challenging structures of macromolecular complexes were solved and the structure-function relationships of different biological systems explored. The results of the studies on the two main systems, LdcI-RavA-ViaA and MCIA, gave rise to original models of their molecular function and opened up novel research directions, currently pursued by us and by others. The cryo-ET structure of the E. coli core signalling unit chemosensory array is an important milestone for the community of researchers who work on bacterial chemotaxis. The minicells developped and used to produce this structure are being shared with labs accross the world for other cryo-ET applications. The developped cryo-ET software tools are publicly available. The collaborative https://teamtomo.org/ platform is operational.