The genes for two types of α2M are present in bacterial genomes : type 1, which contains the thioester bond, and type 2 that does not harbor it. Type 1 bacterial α2Ma persistently co-occur within the same operon with a gene that encodes a cell wall biosynthesis enzyme, Penicillin-Binding Protein 1c (PBP1c). This suggests that the association between the two proteins could be highly advantageous for the cell during infection/colonization, when the outer cell wall is targeted by host defenses. In this situation, α2M and PBP1c could exert the role of ‘guardians of the periplasm’, with PBP1c repairing damaged peptidoglycan, and α2M trapping invading proteases.

The aim of this work was to demonstrate the existence of such complex and characterize this interaction structurally and functionally. For this purpose, α2M and PBP1c from E. coli were studied. The proteins were expressed and purified separately. α2M (also called ECAM in E. coli) is a highly soluble, monomeric protein with a mass of 182 kDa, monodisperse and stable during the course of time. PBP1c is a membrane-bound, 87 kDa protein, predominantly present as a dimer. The complex, reconstituted in vitro by mixing and incubating the proteins for 2 hours, resulted in formation of a complex, demonstrated by appearance of a new peak in size exclusion chromatography. This result was further confirmed by SDS-PAGE, analytical centrifugation and small-angle x-ray scattering (SAXS) experiments. ECAM and PBP1c associated with 2:2 and 4:4 stoichiometries. The activity test confirmed that PBP1c performs polymerization of glycan chains and that its activity is enhanced in the presence of ECAM.

Contact : ibs.seminaires@ibs.fr