Awardees

The severe acute respiratory syndrome coronavirus (SARS-CoV/CoV-2) genome encodes 16 non-structural proteins (nsps), which coordinate cell remodeling, virus replication and participate in viral evasion. Notably, nsp3 contains a protein module termed Macro domain, which carries IFN antagonist activity that interferes with host innate immunity response. This domain is able to bind and hydrolyze ADP-ribose derivatives. This activity is correlated to viral escape and thus makes Macro domains a valuable therapeutic target. In the present paper, we report a SARS-CoV Macro domain structure in complex with a MOPS molecule. Based on our structural data, molecular docking was performed on a set of MOPS analogs in the ADP-ribose binding pocket. We present an ELISA-based assay to select hits based on the inhibition of recombinant SARS-CoV/CoV-2 Macro domain-ADP-ribose complex formation. Among the tested analogs, MOPSO and CAPSO are the more efficient in inhibiting ADP-ribose-binding. Structural analysis of these molecules in the ADP-ribose pocket reveals potential interactions with amino acid residues involved in the coordination of ADP-ribose. Overall, these findings suggest that MOPSO and CAPSO bear potential to be used as a scaffold for the design of Macro domain-specific inhibitors.

A new tool coming from ^nanobodiesR has emerged: ^MegabodiesR.

Megabodies ^R are constructed by grafting a nanobody to a scaffold protein, enhancing the molecular weight while retaining the affinity and specificity of binding towards the target.

The purpose of those megabodies is to overcome two major obstacles that limit cryo-EM resolution: size particles and preferential orientation at the water-air interface (Uchański et al., 2021).

This project aims at developing a methodology to design ^R megabodies from existingR ^nanobodies.

Abstract:

Cancer is a major health problem and important efforts are devoted to find out novel and effective treatments. Despite the progresses achieved in targeted- and immune-therapies, reality shows that in a clinical setting at least one potent cytotoxic compound is required in the therapeutic regimen. Metal-based chemotherapeutics (e.g. cisplatin, carboplatin and oxaliplatin) have shown high clinical relevance becoming first-line treatment for several cancer types. However, the non-physiological nature of the metal often leads to undesired side-effects. This situation has awaked a quest for the development of transition biometal-based therapeutics.

In this project we are developing cytotoxic redox-active Cu complexes and deciphering their location and mechanisms of action inside cells to improve their therapeutic efficiency.

Abstract:

Magnetotactic bacteria (MTBs) are microaerophilic aquatic bacteria that can swim (using their flagella) toward their optimal development zone, the oxic-anoxic transition zone (OATZ). For that, they are helped by magnetotaxis (detection of the Earth's magnetic field) and chemotaxis (detection of the oxygen gradient). These three components (magnetotaxis, chemotaxis, and swimming) define a unique property of MTBs: the magneto-aerotaxis. We demonstrated that the flagellin (the protein that forms the flagellar filament) of MTBs is glycosylated, and that this glycosylation is crucial for the set-up of the filament and therefore for the motility. The synergy of our respective expertise in bacterial genetics, protein crystallography, and glycobiology bioinformatics has allowed us to identify three new types of bacterial glycosyltransferases found in MTBs, as well as in many other bacterial species, and to demonstrate their essential roles in the mechanism of forming a functional flagellum.

Key words:

Magnetotactic bacteria, motility, flagella structure, glycosylation, glycosyltransferases

Abstract:

Filamentous phages are viruses that behave like bacterial symbionts that are an underestimated component of microbiota. They notably improve antibiotic resistance and virulence in human and plant pathogens. Yet, the infection process remains poorly understood at the molecular level, and the dynamic parameters of the process are unknown, as real-time visualization of viral infection was not yet available. In this project, we combined our expertise in the biology of filamentous phages and single cell fluorescent microscopy to decipher the dynamic of the infection process for the fd/E. coli couple. We validated individual fluorescent labeling strategies on the different macromolecular structures involved: the phage capsid, the pilus and the phage DNA and started the monitoring their dynamics during filamentous phage infection at the cell level. We expect these tools to be helpful for structural and biochemical characterization of key infection steps, and to open up prospects for future studies of other filamentous phages associated with pathogens.

Key words:

Filamentous phages, infection, dynamics, single cell microscopy

Redox enzymes are at the heart of a wide range of bioenergetic processes essential for all living organisms. They operate under mild conditions with high specificity, low overpotential and high turnover rates. Deciphering "how redox enzymes operate" is a prerequisite before harnessing their outstanding properties. Amongst a plethora of biophysical techniques, Surface Enhanced InfraRed Absorption (SEIRA) spectroelectrochemistry shines at probing redox enzymes in situ and in operando revealing information on secondary structure elements, orientation, catalytic mechanisms, redox chemistry and functionality.

However, despite this outstanding potential, SEIRA spectroelectrochemistry faces limitations mainly arising from the nature of the biological sample itself. In fact, it is nearly impossible to rely exclusively on intrinsic infrared markers of the protein to elucidate structural or environmental variations at a local level. Consequently, evaluating the immobilised redox enzyme orientation or observing protein dynamics is difficult as it is hampered by the global response of the protein amide bands. Similarly, monitoring catalysis at the redox active site is restricted to only a handful of enzymes.

The VIPER project aims at lifting off those limitations for: (1) improving SEIRA resolution and (2) broadening the range of investigable redox enzymes. The methodology will rely on the introduction of site-specific Vibrational Probes (VPs), into a model redox enzyme (Laccase). Vibrational probes are small molecules which vibrational mode is sensitive to its immediate environment in terms of electrostatics and H-bonding. Laccase catalytic activity couples the one-electron oxidation of a wide range of organic and inorganic substrates to the four-electron reduction of molecular oxygen into water. We will decipher the orientation - activity relationships of laccase upon immobilisation onto electrodes. We will subsequently monitor its structural dynamics at a localised level and ultimately probe the various states of its catalytic cycle. This interdisciplinary project will pave the way for the spectroelectrochemical characterisation of poorly or yet uncharacterised redox enzymes.

Abstract:

Apoptosis is a tightly regulated form of programmed cell death essential for development and tissue homeostasis. A key event in this process is the permeabilization of the mitochondrial outer membrane, driven by the pro-apoptotic BCL-2 family of proteins; Bax and Bak. VDAC, the major channel of the mitochondrial outer membrane, has long been proposed to play a central role in apoptosis through interactions with BCL-2 family proteins. However, despite extensive cellular studies, the molecular basis of these interactions remains poorly understood, and direct structural and biochemical characterization of VDAC-Bax complexes is lacking. Here, we address these questions by a structure-function in vitro approach. We produced and purified the VDAC2-Bax complex in a minimal membrane system. Using complementary biochemical, biophysical, and structural approaches, we demonstrate that Bax directly forms a stable complex with VDAC2. We identify the C-terminal helix α9 as a key determinant of the interaction and show that VDAC2-associated Bax adopts a membrane-embedded, 'primed' conformation in which the N-terminal region and BH3 domain remain accessible. Finally, we establish the specificity of this interaction for the isoform VDAC2 over VDAC1 and propose a mechanistic framework by which VDAC2 defines a distinct intermediate in the Bax activation pathway.

Co-supervisors :

• Lucie BERGDOLL (LISM)
• Guillaume GERBAUD (BIP)
• James STURGIS (LISM)

Summary:

The development of renewable energy sources is of paramount importance in meeting the energy challenge of the ^21st century. Molecular hydrogen's ability to store and transport energy makes it an ideal energy carrier. It is at the heart of research aimed at replacing fossil fuels with responsible, environmentally-friendly alternatives. However, its production method generates significant carbon dioxide emissions, hindering its large-scale integration. A more environmentally-friendly alternative would be to produce it by electrolysis of water, although this method remains costly due to the materials used, which are generally platinum- and iridium-based. Current research is aimed at replacing these expensive noble metals with affordable, sustainable alternatives. The aim of this thesis was to develop molecular catalysts based on non-noble and abundant metals for hydrogen production under mild environmental conditions. The approach taken was to draw inspiration from hydrogenases, biological systems capable of reversibly converting hydrogen, to effectively reproduce natural processes in artificial systems.

Thesis work thus focused on the design of original bio-inspired catalysts, based on non-innocent thiosemicarbazone ligands, with the aim of understanding the impact of their structure on catalytic properties. By playing on the nature and position of the substituents decorating the thiosemicarbazone ligand, several metal complexes, based on nickel and cobalt, were prepared and evaluated for their ability to promote the electrocatalytic reduction of protons to hydrogen. Our studies were also extended to the exploration of different experimental conditions, including the choice of proton source and solvent, with the aim of identifying the most favorable parameters for hydrogen production. This work has enabled us to gain a better understanding of the relationship between molecular structure and catalytic activity, and to identify promising combinations for the development of high-performance molecular catalysts for the electrocatalytic conversion of protons into hydrogen. Our results pave the way for the integration of these catalysts in solid matrices to improve their performance, stability and durability in electrocatalytic hydrogen production.

Co-supervisors :

• Maylis ORIO (iSm2)
• Renaud HARDRE (iSm2)

Abstract:

Bacteria have evolved numerous specialized multiprotein membrane nanomachines to interact with their environment. Among them, Type IV Filaments (TFF) superfamily has emerged as the "Swiss Army knife" of surface molecular machines, capable of mediating an array of cellular functions, including protein secretion, DNA uptake, host-cell adhesion, biofilm formation, prey killing, sensing and various forms of motility.

TFF are ubiquitous nanomachines essential for bacterial survival, however the mechanism triggering active pili synthesis remains unknown. Using a combination of in vivo single-cell analyses and high-resolution NMR spectroscopy, we showed that PilM (a cytoplasmic component of the machinery) consists of a molecular gate requiring a lock and key mechanism to insert the PilB extension motor and activate TFF machineries.

Using two evolutionary distant model systems, Myxococcus xanthus and Vibrio cholerae, we demonstrated that distinct activators, respectively the SgmX protein and the secondary messenger c-di-GMP, both act on the same binding interface situated in the N1D subdomain of PilB to facilitate its interaction with PilM.

Our findings reveal a conserved regulatory hub for TFF machineries, explaining how these nanomachines were co-opted into diverse cellular processes across evolution.

Co-supervisors :

• Romain MERCIER (LCB)
• Latifa ELANTAK (LISM)

Abstract:

This work aims to understand how the_CO2 fixation is driven by the reducing power, in bacteria assimilating carbon by the reverse Krebs cycle. This cycle is powered by low potential ferredoxins, which are reduced by energy sources with a higher redox potential. Deciphering how carbon fixation is linked to energy metabolism in Aquifex aeolicus, an hyperthermophilic bacterium, and identifying enzymes that can bypass this thermodynamic bias was the objective of this project. Using biochemistry, we identified a putative enzyme that uses the electron bifurcation mechanism to reduce low potential ferredoxins from NADPH. This work has made also possible to undertake the essential task of developing the CrispR-Cas9 tool in a hyperthermophilic bacterium which is particularly exciting. This will be used to study the role of genes in vivo not only as part of this project but also to open up the field of possibilities for research on this organism in general.

Key words:

Bacteria, Carbon assimilation, Energy metabolism, Low-potential Ferredoxin, CrispR-Cas9