Structural and biochemical study of the proteins AmiC, NlpD and FtsW involved in the bacterial cell division

Cell division in the Gram negative bacterium Escherichia coli is a highly coordinated mechanism involving various physiological functions such as chromosome segregation, cell envelope invagination, peptidoglycan synthesis at the division site and separation of the daughter cells. All these functions require a high level of spatio-temporal regulation in order to preserve the physical integrity of the cell. At least 20 proteins required for a proper cell division are recruited to the division site to form a supramolecular complex called the divisome. This thesis work focused on three major components of the E. coli division machinery: the N-acetylmuramyl L-alanine amidase AmiC, the LytM factor NlpD and the lipid II flippase FtsW. These proteins are recruited at midcell at a late stage of cell division. FtsW is an integral membrane protein crucial for the translocation of the peptidoglycan precursor from the cytoplasm to the periplasm where it will be processed to produce septal peptidoglycan. AmiC acts as a septal peptidoglycan hydrolase that allow the separation of the daughter cells. This enzyme has been shown to be activated by the LytM factor NlpD.
The crystal structure of AmiC from E. coli presented in this work confirms the presence of an inhibitory helix in the active site. The AmiC variant lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. Furthermore, the direct interaction between AmiC and NlpD has been detected by microscale thermophoresis with an apparent Kd of about 13 µM. The crystal structure of AmiC also reveals the β-sandwich fold of the AMIN domain, responsible for the septal targeting of AmiC to the division site. The two symmetrical four-stranded β-sheets exhibit highly conserved motifs on the two outer faces. Along with the peptidoglycan binding capacity of the AMIN domain, results obtained so far suggest that the AMIN domain could be involved in the recognition of a specific peptidoglycan architecture or a composition different than the lateral peptidoglycan.
Production screenings of FtsW from different strains were realized and FstW from E. coli was purified. This challenging project will require additional efforts to obtain sufficient amount of protein for structural investigation.
Information gathered in this work confirms the high level of regulation of the hydrolytic activity at the septum and gives a structural basis for a more precise molecular characterization of the division site targeting. Disruption or over-activation of these regulation mechanisms could represent a new strategy in the development of antibacterial compounds.