RaftsStruc · Structural sudies of the bacterial lipid rafts.

Structural sudies of the bacterial lipid rafts.

RaftsStruc MSCA IF project was focused on the structural characterization of the lipids rafts proteins by state-of-the-art technique: cryo-electron microscopy. Special focus was put on the components of the lipid rafts involved in S.aureus pathogenesis: penicillin binding protein 2a and its interacting partners, among them Flotilin- the scaffold protein of the Functional Membrane Microdomains (FMM). Infection with methicillin-resistant S. aureus (MRSA) is a global health problem and a cause of high morbidity and mortality (Chambers EH et al. Postgrad. Med.2001). This is due to the great resistance of this bacterium to β-lactam antibiotics, due to the presence of an additional penicillin-binding protein (Pbp2a) (Chambers EH et al. Postgrad. Med.2001). Like other PBP proteins, Pbp2a catalyses the formation of peptide crosslinks between glycan chains of the cell wall. β-lactams act as substrate analogues. When bound to PBP, they inhibit their function; as a result the cell wall is weakened and the cell eventually dies (Chambers EH et al. Postgrad. Med.2001). Pbp2a has very low affinity for β-lactams; it therefore remains active and allows cell growth in the presence of the antibiotics. Pbp2a is a membrane protein and in S. aureus is part of the protein cargo of the FMM and interacts with flotillin A (FloA). The scaffold protein FloA was recently shown to influence Pbp2a oligomerization and/or protein-protein interactions, within lipid rafts, thus affects its function (Garcia-Fernandez et al. Cell, 2017). Yet the mechanism and structural character of this interaction is unknown. The GENERAL OBJECTIVE of this proposal: 1. Obtain a structure by cryo-electron microscopy (cryo-EM) of in vitro-assembled lipid rafts In order to obtain a structure of the in vitro assembled lipid rafts it is important to understand the architecture of the lipid rafts components. We focused on the structure of the lipid rafts protein components also to understand their function: Pbp2a, FloA, SA1401. To achieve that we overexpressed and purified each protein for structural studies also we studied protein-protein interactions and complex formation conditions. 2. Structural study of the interaction between the raft scaffold proteins FloA and Pbp2a (a S. aureus protein responsible for methicillin-resistant S. aureus (MRSA)) by cryo-EM and single-particle 3D reconstruction This objective as a part of objective 2) also was based on overexpression and purification of the full length proteins using detergent based methods and affinity purification. Purified proteins were mixed and analysed for complex formation by Size Exclusion Chromatography and glycerol gradients with and without crosslinking. The samples were analysed by negative staining EM. Unfortunately the stability and quality of the sample was not good enough for further structural studies. However a novel interesting discovery was made: dimerisation of the Pbp2a and its implication on the antibiotic resistance. And we proceeded with the cryo-EM analysis of the dimeric Pbp2a.

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