Stereo views of the AbOmpA-PD complex with DAP and superposed OmpA-like domains. A) Stereo representation of the closeup of the interaction between DAP (represented by its electron density in the omit map contoured at 2σ and represented in the model as the yellow capped stick within) and OmpA. Important residues within this binding pocket are rendered as capped sticks. Hydrogen bonds are indicated as dashed black lines. B) OmpA-like domains of AbOmpA, NmRmpM (PDB code 1R1M), EcPal (PDB code 1OAP), and HpMotB (PDB code 3CYQ) are superposed in the stereo ribbon depiction and shown in green, yellow, orange, and cyan, respectively. Strictly conserved Arg286 and Asp271 within this space are rendered as capped sticks.

PGN pulldown and ITC binding assays of meso-DAP with AbOmpA-PD. A) PGN pulldown results. B–D) ITC results with meso-DAP (K_d 2.2 μM; B), compound 2 (K_d 3.2 μM; C), and compound 3 (D).

Omit map (contoured at 2σ) of AbOmpA-PD (green) complexed with compound 5 (yellow).

Sequence alignment of OmpA-like domains and the crystal structure of AbOmpA-PD. A) Multiple amino acid sequence alignment of AbOmpA homologs. Secondary structure elements from the AbOmpA structure are indicated above the alignment. Residue numbers are for AbOmpA, and red blocks denote regions of sequence identity across all homologs. Partially conserved residues are boxed. Residues important for DAP binding by AbOmpA are highlighted with stars below the sequence alignment. PaOprF, Pseudomonas aeruginosa OprF; CjCadF, Campylobacter jejuni CadF; EcPal, E. coli Pal; HiPal, H. influenzae; PaOprL, P. aeruginosa Pal; HpMotB, H. pylori MotB; VaMotY, Vibrio alginolyticus MotY; EcSciZ, E. coli SciZ. B) Ribbon depiction of AbOmpA-PD in complex with DAP. Model was rendered on the basis of its secondary structure, in which α helices and β strands are shown in red and yellow, respectively. Structure of bound DAP is drawn as a space-filled model, and atoms are colored based on atom type (nitrogen, blue; oxygen, red; carbon, gray). C) Surface electrostatic potential depiction of AbOmpA (red, negative; blue, positive). Residues responsible for the basic patches that would interact with the PGN on the surface are indicated by the dotted area.

Chemical structures of compounds used in this study. Compound 1, DAP; compound 2, tripeptide DAP; compound 3, tripeptide Lys; compound 4, NAM-tripeptide; compound 5, NAM-pentapeptide.

Chemical shift perturbations of AbOmpA-PD with various PGN derivatives and the computational model of the AbOmpA-PD complex with the PGN ligand. A) Differences in the ¹H-¹⁵N chemical shift (Δδ={δH²+0.2×(δN²)}^1/2) of DAP-bound AbOmpA-PD from those of the protein bound to compound 2 (left panel), compound 4 (middle panel), and compound 5 (right panel). Colors magenta and orange represent large (Δδ>0.1 ppm) and moderate (0.1>Δδ>0.05 ppm) changes in the chemical shifts, respectively. B, C) Stereo images of the computational model of PGN binding to AbOmpA-PD, represented using a ribbon model (B) and a solvent-accessible Connolly surface (C). PGN ligand is rendered as capped sticks; peptides are in blue; tetrasaccharide backbone is orange. Cavity that accommodates the side chain of DAP is at 6 o'clock in relation to the images in B and C.