Missions

The non-canonical 3',5'-cyclic nucleotide monophosphates (cNMPs) cCMP and cUMP have recently been recognized as novel second messengers in bacteria, where they activate immune pathways against phage infection. Their presence has also been firmrly demonstrated in diverse eukaryotic systems, although their molecular targets and physiological roles remain enigmatic. This international ANR/DFG-funded project aims to contribute to elucidate, at the molecular scale, how and where cyclic UMP and CMP are generated, regulated, and integrated into signaling networks. The recruited postdoctoral fellow will characterize at the biochemical and structural levels the enzymatic specificities of nucleotidyl cyclase (NC) enzymes that produce cCMP and cUMP, and investigate the regulatory mechanisms of cUMP/cCMP-dependent effectors. Two distinct bacterial NC families will be investigated: ExoY toxins, which target and use eukaryotic actin monomers or filaments (as cofactor) for their activation inside host cells, lead to supraphysiological levels of both purine and pyrimidine cNMPs; and pyrimidine cyclases (PycC), which generate cUMP/cCMP as part of bacterial antiviral defense. To support and extend the enzymatic and structural investigations and to explore strategies for inhibiting ExoY NC toxins, the candidate will optimize a high-throughput real-time fluorometric assay to monitor and characterize NC activity in vitro under diverse conditions. Structural studies will integrate protein engineering with X-ray crystallography, complemented by cryo-EM to resolve large and dynamic protein assemblies that are challenging to crystallize. Informed by structural insights into cNMP-dependent effectors, the candidate will help design and refine innovative fluorescent biosensors for precise monitoring of cNMP levels in vitro. These combined approaches will provide the tools to follow, over the long term, how purine and pyrimidine cNMP concentrations change inside living cells, and to uncover the signaling targets and pathways that pyrimidine cNMPs control.

Activités

The successful candidate will play a central role in combining biochemical, biophysical, and structural approaches to address fundamental questions in nucleotide signaling. Main activities will include: (i) expression and purification of recombinant proteins in E. coli, covering soluble and/or membrane-associated targets, to enable in-depth biochemical and structural studies; (ii) investigation of protein–protein and protein–ligand interactions using assays established in the team (e.g., pyrene-actin polymerization, cosedimentation) and advanced biophysical techniques available on institute platforms, including isothermal titration calorimetry, microscale thermophoresis or SECMALS; (iii) optimization of high-throughput real-time fluorescent assays in microplate formats to monitor NC activity and systematically assess cofactor dependencies, activation mechanisms, and substrate specificities; (iv) application of structural biology methods, such as protein crystallization (standard/LCP), X-ray diffraction, EM negative staining, and cryo-EM to study dynamic states and large protein assemblies that are difficult to crystallize; (v) design and implementation of molecular biology strategies, such as targeted mutagenesis or the construction of fluorescent protein fusions, to probe cNMP concentrations in vitro in fluorescence microplate screening assays; (vi) contribution to the development and optimization of FRET-based biosensors to monitor specific cNMP concentrations in vitro, guided by structural insights into cNMP-dependent effectors; (vii) rigorous data analysis, interpretation, and clear presentation of results in internal meetings and within the collaborative network; and (viii) active participation in the dissemination and valorization of results through reports, manuscripts, and presentations at national or international meetings. The position offers access to state-of-the-art cryo-EM facilities at I2BC and SOLEIL synchrotron, and provides opportunities to interact with French and international collaborators in biochemistry, microbiology, cell biology, proteomics, and pharmacology, ensuring a rich interdisciplinary training environment.

Compétences

Applicants must hold a PhD in biochemistry, biophysics, structural biology, or a related field, with solid experience in molecular cloning, bacterial expression, and purification of recombinant proteins. Prior experience in one or more of the following areas will be considered as strong asset: FRET/BRET fluorescence-based assays, cryo-electron microscopy (single particle analysis), and/or membrane protein purification and structural characterization. A strong interest in structural biology, combined with familiarity with in vitro experimental design and data analysis, is essential. The project particularly welcomes candidates with a demonstrated interest in structural enzymology, and/or the development of innovative fluorescence-based tools (FRET, BRET). Beyond technical expertise, excellent communication skills in English, both written and spoken, are required to interact effectively within the international collaborative network and to contribute to presentations and publications. The ideal candidate will combine autonomy, rigor, and creativity in designing and performing biochemical, biophysical, and structural experiments, while also valuing teamwork and scientific exchange. We seek a colleague who enjoys sharing results and ideas openly and contributing to the synergy of a small research group, rather than working in isolation. Strong interpersonal skills, openness to feedback, and adaptability are essential, as is the ability to document, interpret, and present results clearly. A strong motivation to explore novel aspects of nucleotide signaling and to engage with complementary expertise in microbiology, cell biology, and pharmacology will be highly valued.

Contexte de travail

The candidate (M/F) will work in the team “Structural Biochemistry of Microtubules, Kinesins and their Cargos” in the the Institute of Integrative Biology of the Cell (I2BC, about 600 persons), University Paris-Saclay, CNRS, CEA (www.i2bc.paris-saclay.fr/institute/), located at Gif-Sur-Yvette in Paris-South at 45 min from Paris by local train public transport (RER B). The institute is well equipped for biochemical and structural studies, with Platforms for Interactions of Macromolecules, crystallization robots, and a cryo-electron microscopy facility included two cryo-electron microscopes 200 kV (FEG) and 120 kV (Tecnai FEI). The team has regular access to SOLEIL synchrotron for X-ray and very recently cryoEM (300 kV , Titan Krios G4) data collections. The structural biology team's website is: https://www.i2bc.paris-saclay.fr/structural-biochemistry-of-microtubules-kinesins-and-their-cargos-gigant-menetrey/
The project is carried out within a stimulating international collaborative network supported by the National Research Agency (ANR) and DFG (german agency) (ANR/DFG International Collaborative Research Project), providing interdisciplinary exchanges with collaborators in microbiology, cell biology, proteomics, pharmacology and host–pathogen research. The position may be extended for several months, depending on project needs and the availability of allocated funding, subject to approval of a funding extension.

The candidate (M/F) will benefit from:
• Access to the laboratory via RER B, which connects Paris and the Parisian suburbs (~45 min Paris center-Gif-Sur-Yvette by RER B).
• Transport benefits: possible partial coverage of transportation costs, depending on condition
• Access to an on-site collective subsidized restaurant on campus
• Social offers: participation in health insurance, local Social Action and Solidarity Committee (sports, leisure, ticketing, etc.)
• Working hours: 38.5 hours per week, 44 days of annual leave per year.

Contraintes et risques

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