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.

Cationic antimicrobial peptides (AMPs) that can target multidrug-resistant pathogenic bacteria via multiple mechanisms are considered promising alternatives to antibiotics. Small (~9 kDa) highly acidic acyl carrier proteins (ACPs), which are a well-known cofactor protein in bacterial fatty acid synthesis (FAS), are a potential intracellular target for AMPs. A previous study has demonstrated that the human AMP LL-37 can bind to ACP and thereby affect FAS and the bacterial membrane integrity. In this work, we have investigated the interactions of different classes of AMPs and antibiofilm peptides (ABPs) with the ACPs of two pathogens. We first studied the folding characteristics of the two ACPs and found that Pseudomonas aeruginosa ACP (PaACP) is fully folded at neutral pH in the absence of divalent cations. On the other hand, the homologous Francisella novicida ACP (FnACP) is unfolded at low ionic strength, but it adopts a fully folded conformation after the addition of divalent cations such as Ca²⁺ or Mg²⁺. These distinct characteristics were shown to be related to a unique His residue that is involved in a stabilizing cation–π interaction. Subsequent biophysical SPR and NMR interaction studies reveal that cationic AMPs and ABPs such as LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B and IDR-1018, but not F5W-magainin 2, can bind to both apo- and holo-ACPs. Binding of Arg-rich peptides is preferred over their Lys-rich analogs. Interestingly, all the peptides bind to holo-ACP with higher affinity than to apo-ACP, which lacks the functionally important phosphopantothenate group. NMR peak intensity perturbation data reveal that helix II of ACP, which is known to be directly involved in complex formation with bacterial FAS enzymes, acts as a common and main recognition site for the peptides. We propose that binding of AMPs and ABPs to this region of bacterial ACPs can directly block fatty acid synthesis and interfere in other ACP-dependent biosynthetic and regulatory events, which in turn could contribute to killing the bacteria and could also intervene in biofilm formation.