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PhD in molecular simulation of biological systems

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Français - Anglais

Date Limite Candidature : jeudi 14 juillet 2022

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General information

Reference : UMR8000-FABCAI-001
Workplace : ORSAY
Date of publication : Thursday, June 23, 2022
Scientific Responsible name : Fabien Cailliez
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 3 October 2022
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly

Description of the thesis topic

NADPH oxidases (NOX) are enzymes playing a crucial role in the defense of many organisms (mammals, fungi, plants…) towards pathogens (Nauseef, Curr. Opin. Immunol. 60, 130–140 (2019)). They catalyze the reduction of oxygen to produce reactive oxigen species, called ROS (superoxide ions or hydrogen peroxide depending on the NOX type). Their malfunction (under- or over-production of ROS) leads to pathologies such as oxidative-stress-related ageing, carcinogenesis, neurodegenerative diseases and inflammatory mechanisms. The flavocytochrome b558 (Cytb558) lies at the heart of the machinery of NADPH-oxidases of leukocytes. It is composed of 2 transmembrane subunits: NOX2 and p22phox. The crystallographic structure of NOX5 (isoform of NOX2) has revealed the location of the redox cofactors that carry temporarily the electrons: a flavin and two hemes (Magnani et al., Proc. Natl. Acad. Sci. 114, 6764–6769 (2017)). The exact mechanism of how NOXs regulate electron transfers to produce superoxyde anion is however still largely unknown.

Molecular simulation is a powerful tool to get access to molecular mechanisms of biochemical processes. In the context of NOX, we have recently revealed original mechanistic details on inter-heme ET and on internal cavities allowing oxygen binding to the transmembrane domain (Wu et al., Front. Chem. 9:650651 (2021)). Many questions are still unanswered, such as:
• Are the 2 electrons brought by NADPH transferred one-at-a-time to successively reduce 2 dioxygen molecules, or are they loaded in the membrane (one on each heme) prior to dioxygen binding?
• Is the reduction of the external heme necessary for the binding of oxygen to the protein?
• Which electron transfer limits the speed of the full ET process in NOX?
• Which are the key residues that control each ET step?

The goal of this PhD project is to use molecular simulation to characterize the electron transfer steps (NADPH → flavin → Heme 1 → Heme 2 → O2) in NOX that lead to the production of superoxide anions. To this end, a large variety of molecular modelling methods are necessary, from the building of 3D structures of transmembrane protein complexes using bioinformatic tools to quantum chemistry calculations, and a large use of classical molecular dynamics simulations using advanced forcefields. The main aspects that will be developed during the project are the following:
1. Estimation of the thermodynamics of electron transfer steps. This task will be performed using dedicated protocol that are mastered in our group (de la Lande et al., Archives of Biochemistry and Biophysics 582, 28–41 (2015); Cailliez et al., J. Am. Chem. Soc. 138, 1904–1915 (2016)). It consists in running molecular dynamics simulations of the system in various redox states corresponding to intermediary states, associated with quantum chemistry calculations. The use of an advanced forcefield with a careful description of electrostatic interactions is mandatory to achieve a good accuracy in the calculations. The polarizable and multipolar forcefield AMOEBA will be used for that purpose. Some forcefield parameters of the cofactors in their different redox states will have to be calibrated and validated in the course of the study.
2. Evaluation of rate constants of electron transfer steps. The Marcus theory formalism allows to estimate ET rate constants from thermodynamic parameters and coupling between electronic states. The calculation of the latter requires the use of specific quantum chemistry methodologies implemented in the DFT software deMon2k by our group at Institut de Chimie Physique (ICP).
3. Identification of key residues for ET steps. Thermodynamic and kinetic information obtained in steps 1 and 2 will allow to detect the implication of key aminoacids. In silico mutations will be performed to (in)validate those hypotheses with the aim to ultimately propose an entire mechanism of electron transfer steps in NOX2.
4. Diffusion of oxygen and superoxide anion. The final step of NOX2 functioning is the reduction of dioxygen into superoxide anion. It is not clear whether this step is limited by the electron transfer or by diffusion of the oxygenated species. Thus, the study of the diffusion and binding of oxygen and superoxyde anion will be performed with the use of molecular dynamics simulations.

This PhD project will be part of a collaboration between theoreticians (at ICP and at LBT, in Paris) and experimentalists (at ICP and I2BC in Gif-sur-Yvette) aiming at the characterization of all the ET steps that lead to the formation of superoxyde anion and a better understanding of NOX functioning. Results that will be obtained from molecular simulation will thus be correlated with experimental data and stimulating experience/theory discussions will enrich the project.

Work Context

The project will take place at the Institut de Chimie Physique (CNRS UMR 8000) of Paris-Saclay University under the supervision of Dr. Aurélien de la Lande and Dr. Fabien Cailliez. The PhD candidate will be a member of the 2MIB Doctoral School of Paris-Saclay University.
During the thesis, there will be strong interactions with members of LBT in Paris (Dr. Marc Baaden, Dr. Sophie Sacquin-Mora, Dr. Antoine Taly), experts in the simulation of transmembrane systems.

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