NMR structural analysis and peptidoglycan binding properties of the peptidoglycan associated lipoprotein (PAL) from Escherichia coli

The peptidoglycan associated lipoprotein (Pal) is crucial in Gram-negative bacteria, participating in outer-membrane integrity and septal wall constriction during cell division. It is also implicated in pathogenesis by mediating sepsis and immune responses. Pal has been one of the most intensively studied vaccine targets and herein we report a structural characterization and functional analysis of Escherichia coli Pal (Ec.Pal), as a member of the peptidoglycan-binding protein (PGBP) family. Multidimensional solution NMR spectroscopy was employed to obtain backbone assignments for truncated and full-length constructs of Ec.Pal, revealing that these proteins adopt the characteristic secondary structure of the OmpA_C-like domain and that the core residues fold similarly to the crystal structure reported for a truncated protein (PDB 1OAP). However, full-length Ec.Pal possesses a previously unobserved N-terminal α1-helix, which in conjunction with a 30-residue flexible N-terminal linker, distinguishes Ec.Pal from other PGBP members. Biophysical studies further demonstrated the role of this terminal region in mediating the dimerization of Ec.Pal, contrasting its behavior with other PGBPs. Moreover, our findings for an acylated version of Ec.Pal which was purified as a SMALP-complex, suggest that Ec.Pal can interact with membrane mimetics through the flexible N-terminal region as well. Additionally, the C-terminal domain of Ec.Pal was shown to bind peptidoglycan (PG) components and co-purify with the PG-precursor (PGp), highlighting its role in cell wall dynamics. These results contribute to understanding the structural basis of Ec.Pal's function in bacterial membrane biology and its potential as a therapeutic target.