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/(si apre in una nuova finestra) 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.
