Structure and Stability of Integral Membrane Protein and Phage Assemblies Team (Cécile Breyton)

Team Leader: Cécile BREYTON (DR CNRS)

Current members
Cécile Breyton (DR - CNRS)
Christine Ebel (DR - CNRS)
Claudine Darnault (Tech - CEA)
Corinne Deniaud (Ing - CEA)
Aline Le Roy (Tech - CEA)
Alessio D’Acapito (PhD student)
Alice Decombe (Post-doc)
Rubén Perez-Bucio (M2)

Expertise

Biochemistry of membrane proteins; Biophysics; Crystallography; Small angle X-ray and neutron scattering; Cryoelectron microscopy.

Main Research themes

The structural investigation of the first step of phage infection in the phage T5 – E. coli system.
60% of known phages, viruses that attack bacteria, consist of a capsid protecting the viral DNA and a long, flexible tail, which tip recognises the host, resulting in the perforation of the bacterial cell wall and the safe delivery of the DNA into its cytoplasm. We aim at deciphering the molecular mechanisms which commits the phage to infection and perforation of the whole cell wall.
In the last years, we have concentrated in understanding the molecular mechanism of E. coli cell-wall perforation by siphophage T5. At the distal extremity of its tail, T5 bears three dispensable L-shaped fibres and a straight fibre, at the tip of which stands the Receptor Binding Protein pb5 (RBPpb5). Binding of the latter to FhuA, an outer-membrane transporter, commits the phage to infection. By electron cryo-microscopy(cryo-EM) and in collaboration with the group of Guy Schoehn, we have determined the structure of the RBPpb5-FhuA complex and of T5 tail tip, before and after interaction with FhuA reconstituted into a nanodisc (figure). This latter detail is important, as it provided a membrane to the phage, resulting in trapping an intermediate of outer-membrane perforation. These structures allowed us to unravel how receptor binding triggers infection and to decipher the molecular pathway that commits the phage to perforating the outer-membrane.

Shortly after infection, T5 produces a small lipoprotein, Llp, directed to the inner leaflet of the outer-membrane, which protects the viral factory from over-infection. We have shown that Llp binds to FhuA, preventing new RBPpb5 binding and we have extensively characterised the Llp-FhuA interaction in vitro and in vivo.

In this context, the team is a member of the Phages.fr thematic network.

The development of methods for the functional and structural studies of membrane proteins.
We have we have studied the opportunity of using various detergent (LMNG), fluorinated surfactants and more recently metallacarboranes for the manipulation, the stabilisation and the crystallisation of membrane protein. Also, we successfully use Small Angle Neutron Scattering (SANS) and Analytical Ultracentrifugation (AUC) in different collaborative projects.

We are evaluating protocols, for the characterization of membrane proteins, using Analytical Ultracentrifugation and Size Exclusion Chromatography coupled to Light Scattering.

Small Angle Neutron Scattering (SANS) is a powerful technique to investigate the solution structure of membrane proteins, in that it allows cancelling the contribution of free micelles and bound detergent. We have used SANS to reveal fast collisional lipid exchange within patches of lipid bilayers stabilised by SMA, an amphipatic polymer used to manipulate membrane proteins. We are studying the conformations of the proteins SpNox and BmrA, in collaboration with F. Fieschi (M&P, IBS), and J.-M. Jault (IBCP, Lyon), respectively, and with A. Martel (ILL).

Keywords

Structure-function relationships of receptors, channels and transporters; Biophysics and biochemistry; Molecular and structural virology; Bacteriophages; Surfactants.

Alumni

(L1, L2, L3 and M1 students are not mentionned)
Rubén Perez-Bucio (M2, 2025)
Estelle Marchal (CDD, 2023-2024, ANR ProMeNix)
Romain Linares (Post-doc, 2017-2022, ANR PerfoBac, ANR ImmunoPhage)
Andromachi Papagianoula (M2, 2022)
Séraphine Degroux (M2 – PhD, 2018-2022, UGA)
Charles Arnaud (M2 – PhD, 2014-2017, Labex Gral)
Swetha Stanley (M2, 2020)
Ilham Bouchemal (CDD, 2018-2020, ANR Fluor)
Nathan Epalle (M2, 2019)
Waqas Javed (PhD, 2016-2020)
Béatrice Schaack (CEA researcher, 2013-2017)
Alice Tissot (M2, 2013)
Flavien Grégoire (M2, 2013)
Kai Wang (M2, 2013)
Virginie Guillon (XX, 2013)
Mathilde Lethier (CDD, 2010-201X, )
Cindy Pereira (M2, 2012)
Ziad Ibrahim (M2, 2011)
Ali Flayhan (M2 – PhD, 2008-2012, ITN SBMP)
Sarah Saab (M2, 2009)
Nathalie Hauet (Post-doc, 2008-2010, ANR PCV-Fluorinated surfactants)

Recent grants

2023-2025 : GRAL, PhD Operating Cost for A. D’Acapito
2022-2026 : ANR, Metallacarborans : an opportunity for the study of membrane proteins
2022-2025 : ANR, Recognizing sweet surfaces – structure and function of a siphovirus infection device at the Gram- bacterial membrane
2020-2023 : ANR, Molecular mechanisms of phage T5 induced immunity
2020-2021 : CEA, Metallacarborans for the study of membrane proteins
2018-2019 : CEA, Mass spec of siphophage T5
2016-2021 : ANR, Fluorinated surfactants for the study of membrane proteins
2016-2020 : ANR, How do siphophages perforate the bacterial cell wall?