Periodic Reporting for period 1 - RespViRALI (Structural and functional insights into the assembly of respiratory complexes by a novel putative chaperone)
Reporting period: 2019-07-01 to 2021-06-30
RavA is an AAA+ ATPase of the MoxR type, a protein family of which the members are suggested to be involved in chaperone functions. RavA is involved in the bacterial acid-stress response by modulating the activity of the acid-stress inducible lysine decarboxylase (LcdI) during stringent response, and is shown to interact with ViaA, an operon partner of RavA containing a Von-Willebrand Factor A (VWA) domain. The rationale for the involvement of LdcI, RavA and ViaA in the assembly of Nuo and Frd is that :
1) RavA and LdcI together form a huge cage-like assembly which is shown to interact with specific subunits of Nuo and Frd.
2) ViaA, a protein of unknown structure proposed to act as a shuttle protein between the LdcI-RavA cage and its substrates, was also shown to interact with Nuo and Frd subunits.
3) E. coli cells lacking ravA/viaA genes acquire resistance to aminoglycosides, a class of antibiotics requiring proton motive force, generated by bacterial respiratory complex 1, for their uptake.
We aimed to elucidate the involvement of the LdcI-RavA-ViaA triad in the assembly and maturation of Nuo and Frd by a combination of biochemical/biophysical methods to validate interactions between LdcI, RavA and ViaA and specific soluble sub-complexes of Nuo and Frd, with a structural characterization of the most promising interactions using an integrated structural biology approach combining low- and high-resolution techniques.
We thus wondered if the previously proposed interactions with Nuo might be mediated by an interaction of RavA and ViaA with specific lipids in the E. coli inner membrane. Dot-blot assays performed by a visiting scientist in the Gutsche group, Dr. Ladislav Bumba, demonstrated that RavA binds to specific lipids present in the E. coli inner membrane. To structurally investigate these RavA/ViaA-lipid interactions, cryoEM grids were prepared containing a mix of RavA or ViaA with liposomes enriched with lipids interacting with RavA or ViaA respectively. We subsequently collected 140 tomographic tilt series of RavA bound to liposomes on the Titan Krios in EMBL Heidelberg. Processing of the data, including alignment of the different tilt series, particle picking and subtomogram averaging, is ongoing. In parallel, 18 tilt series of ViaA-decorated liposomes were collected at the Titan Krios microscope in ESRF, Grenoble.
In addition, given the absence of interactions between LdcI, RavA or ViaA, and the soluble subunits of Nuo/Frd, we focused our efforts on obtaining higher-resolution cryoEM structures of the RavA hexamer and the LdcI-RavA complex, and determining the supramolecular architecture of LdcI filaments at low pH.
The first study, performed in collaboration with Benoit Arragain and Dr. Hélène Malet, demonstrated that the structure of the LdcI-RavA cage is even more intricate than initially described. Inside the cage, RavA hexamers adopt asymmetric spiral conformations containing a gap, or seam, reminiscent of other AAA+ ATPases recently characterized by cryoEM. Surprisingly, the orientation of the nucleotide-free seam in RavA spirals is constrained to only two possible positions. CryoEM structures of free RavA in complex with either ADP or ATPγS , obtained by a PhD student in the lab, Matt Jessop, demonstrate the presence of a spiral state with one seam, as well as a two-seam state, which we propose to correspond to an intermediary step in between switching of the gap to an opposite position in the RavA hexamer. Taken together, our findings suggest a novel mechanism of MoxR ATPase activity in which ATP hydrolysis does not progress sequentially around the ATPase spiral, but switches between opposing positions across the hexamer, thereby linking ATP hydrolysis to mechanical force (Jessop M.*, Arragian B.*, … , Felix J.#, Malet H.# and Gutsche I.#, 2020, Communications Biology 3:46, *:equal contribution, #: co-corresponding author).
The second study, performed in collaboration with the team of Dr. Dominique Bourgeois, investigates the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterization with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Three-dimensional localization of endogenous wild-type LdcI in acid-stressed E. coli cells reveals that it organizes into patches following an apparent long-range pseudo-helical order. Furthermore, we show that in vitro LdcI assembles into filaments at low pH and solve the structure of these filaments by cryoEM, thereby revealing the structural determinants of LdcI polymerization, which we confirm by mutational analysis (Jessop M.*, Liesche C.*, Felix J.*, … and Gutsche I., 2020, bioRxiv preprint under review, *:equal contribution)."
However, the biological role of the RavA-LdcI cage-like complex and ViaA remains unclear. Our results suggest that, rather than directly binding to Nuo and Frd subunits, RavA and ViaA may influence resistance to aminoglycosides through interaction with the inner membrane of E. coli. Structural and functional investigation of the interaction between RavA/ViaA and the E. coli inner membrane by cryo-electron tomography, cellular EM, fluorescence microscopy, mass-spectrometry and phenotypical analysis of mutants deficient in lipid binding is ongoing. We anticipate that these studies will finally shed light on the elusive role of the ViaA-RavA-LdcI protein triad.