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Catching in action a novel bacterial chaperone for respiratory complexes

Periodic Reporting for period 4 - Chap4Resp (Catching in action a novel bacterial chaperone for respiratory complexes)

Reporting period: 2020-04-01 to 2021-09-30

Cellular respiration is a set of biochemical pathways by which cells fuel electrons released from nutrients, through a molecular wire of metal clusters and cofactors embedded in respiratory factories in the inner membrane of mitochondria and bacteria, to oxygen or, in bacteria capable of anaerobic respiration, to an alternate end acceptor. Proton-pumping enzymes of the respiratory chain work like a battery, coupling the flow of electrons to the transfer of protons across the membrane and thereby creating a proton motive force (PMF), used by the ATP synthase to generate the universal energy currency, ATP.
Although respiration is a vital part of metabolism, the battery occasionally leaks and transfers electrons directly to oxygen, producing reactive oxygen species (ROS) such as H2O2, O2- and OH°. These in turn harm the respiratory complexes by releasing iron from their Fe/S clusters, thus producing even more ROS through the Fenton reaction, and damaging DNA, proteins and lipids, causing ageing and diseases. In addition, many drugs and toxins target respiratory complexes to affect their integrity and inhibit their activity. Conversely, defects in bacterial respiratory complexes that hamper their proton pumping action make the bacterial cell resistant to an important class of antibiotics, aminoglycosides, requiring PMF for uptake.
Understanding assembly pathway of the respiratory complexes is therefore an extremely important goal and a particularly challenging one, because of their membrane location, multisubunit composition and cofactor insertion. For example one largest membrane protein assemblies, the respiratory Complex I from the human mitochondria is composed of 45 subunits. The respiratory Complex I from a bacterium Escherichia coli is built by 14 subunits conserved from bacteria to humans, with 9 Fe/S clusters and a flavin, and, although already very intricate, can be therefore considered as a simplified model of its mitochondrial homolog. The objective of this project is to investigate if the huge E. coli macromolecular cage, the structure of which we recently solved by cryo-electron microscopy (cryoEM), in conjunction with a novel protein cofactor, is a specific chaperone for Fe/S cluster biogenesis and assembly of respiratory complexes, and if yes, would is its mechanism of action.
To achieve our goal, we are using an integrated multi-level approach, combining high- and low-resolution structural biology techniques, in vitro biophysical, biochemical and spectroscopic studies, optical imaging and cryogenic correlative light and electron microscopy. In the first years of this project, we created a toolbox of plasmids coding for our proteins of interest with a variety of different tags, expressed these proteins either individually or together, established purification protocols for individual proteins and their potential complexes, and characterized the three individual components of our presumed chaperone system. While we are making major advancements in our understanding of these components, their interplay and their cellular regulation and location, at the moment we cannot confirm any previously published data on interactions of our protein triad with the two major iron-sulfur cluster biogenesis systems of E. coli or with the respiratory complexes. Thus, we started also working on a eucaryotic chaperone triad which is known to interact with the eukaryotic Complex I.
In parallel to analyzing structure-function relationships of our system in vitro, in a purified state, and in vivo at room temperature, we are also trying to go beyond the state of the art and are working on visualisation of internal bacterial structures inside the cell by super-resolution fluorescence microscopy at cryo temperatures and by cryo-electron tomography. Since these studies require many technological and methodological developments, high-end data collection, and sophisticated image analysis, the results may not be visible before a couple of years.