Fig. 6: Molecular basis that underlies HNP1-OmpA interactions.

A The structure of A. baumannii OmpA constructed using AlphaFold 3. The transmembrane domain is in green, the peptidoglycan-binding domain in the periplasm is in yellow, and the two extracellular loops (L1 and L2) are in red. B Binding affinity between the full-length wild-type OmpA (OmpA WT) and HNP1, as well as the OmpA ΔL1L2 mutant (OmpA-ΔL1L2) and HNP1, assessed by fluorescent polarization. C Binding affinity between the transmembrane domain of wild-type OmpA (TM) and HNP1, as well as the OmpA ΔL1L2 mutant (TM-ΔL1L2) and HNP1, assessed by fluorescent polarization. Results of B and C are mean ± SD representative of at least two experiments (n = 2 biological replicates per experiment). D Conformations at different time points of HNP1 and OmpA in molecular dynamics simulation. OmpA is shown in green, interacting HNP1 molecules (M1 and M2) in red, and diffusing HNP1 molecules (M3 and M4) in gray. E The decomposition of individual residue contributions of OmpA in the total binding free energy during 100–1000 ns of MD simulation analyzed using MM-GBSA. F Effect of HNP1 Ala-substitution mutants on biofilm formation characterized by CV staining. Results are mean ± SD of two independent experiments (three parallels per experiment). G Position of critical residues on an HNP1 dimer (PDB: 3GNY). H Restorage of hydrophobicity at position 26 of HNP1 restored its biofilm-enhancing effect. Results are violin plot of four independent experiments (three parallels per experiment). Statistical significances (F and H) were evaluated by one-way ANOVA with Tukey correction for multiple comparison. See also Supplementary Figs. S7–S11 and Supplementary Movie 2.