Five natural darobactins (DA to DE) were previously described. In recent work by our group, the non-natural derivatives D1 to D21 were produced, including a derivative in which the terminal phenylalanine (DA, W1N2W3S4K5S6F7) is exchanged with a tryptophan (D9, W1N2W3S4K5S6W7) as seen in Figure a-b. The latter increases the antibacterial activity 2-to 8fold against a large panel of pathogens. The increased antibacterial activity of modified D9 compared to the native DA was the result of engineering the darobactin biosynthetic pathway and created a basis for new targeted structural improvements. To determine if D9 shares the same binding site as DA, we first resolved the cryo-EM structure of the E. coli BAM complex bound to D9. While the overall average resolution was determined to be 3.4 Å, the local resolution estimated using Cryosparc revealed a resolution of 3.0 Å around the D9 binding region (Figure ; Figure S 1 a-e; Table ). Binding to the β-barrel of the E. coli BAM complex was deciphered revealing high similarities to the binding of DA with the BAM complex shown in Figure d in addition to the overlaid electron density of both compounds (Figure S 2). D9 also binds to the β1 of the lateral gate of BamA with multiple hydrogen bonds in antiparallel β-sheet conformation, mimicking a β-sheet extension thereby blocking the functional region of BamA (Figure ; Figure S 1 f, g). Moreover, the N-and C-terminal ends of D9 are also tightly anchored to BamA via electrostatic and hydrophobic interactions (Figure ). Furthermore, the distances for the establishment of hydrogen bonds of D9 to atoms of the binding site are well within the expected values and suggest that altered core peptides result in highly active darobactin derivatives such as the previously published darobactins DB, D4, D9, D11, D14, and D16. Of note, D9 is more tightly bound to BamA, as shown by comparison with native DA (Figure ), which could be explained by additional side-chain and backbone interactions of L-tryptophan at position 1 with NH of I430, at position 6 with the carbonyl group of N422, and at position 7 with the carbonyl group of S425 (Figure ). The novel hydrogen bond interactions and accompanied changes in D9 orientation are at the expense of L-asparagine hydrogen bond interaction on position 2 with the carbamoyl oxygen of the sidechain (Oδ2) of N427. Remarkably, the Cterminal carboxyl group of the terminal tryptophan forms a shorter hydrogen bond with NHδ2 of N422 in contrast to L-phenylalanine (Figure ). Furthermore, the aromatic group of the C-terminal tryptophan of D9 is significantly extended into the hydrophobic pocket formed by residues F426 and V444, potentially generating strong hydrophobic interactions (Figure ). As a consequence, this might improve the ability to seal the lateral gate compared to native DA. Taken together, the additional interaction sites of D9 result in tighter binding, providing (1) structural evidence for the basis of 8-fold enhanced activity of D9 over DA and (2) proof of concept that additional chemical alterations on DA could further improve binding as well as biological activity. For the design of new non-native darobactin derivatives, we considered currently available data, including relevant interactions of D9 (WNWSKSW) (Figure ) and DA (WNWSKSF) with the BAM complex and the bioactivity ranking of darobactincontaining extracts (DA to DE and 1 to 21), as well as the pure DA, DB and D9. To ensure proper darobactin cyclisation and ligand-receptor interaction (Figure ), positions 1, 2 and 3 of darobactin remained unaltered. In particular, manipulations to amino acid position 2 were avoided as this has previously resulted in reduced production rates and decreased antibacterial activity. Consequently, the binding behaviour of DA and D9 on E. coli BAM complex revealed positions 4, 6 and 7 as the most flexible positions for core sequence engineering, without deteriorating target binding (Figure ). Therefore, we designed fifteen new derivatives with alterations in these positions (Table ): The increased interaction of the C-terminal tryptophan at position 7 compared to smaller phenylalanine motivated the design of D26 to D28, which carry terminal L-histidines. The expectation was that the aromatic imidazole ring of histidine could form strong interactions with the adjacent amino acids of BamA, similar to the aromatic rings of L-phenylalanine or L-tryptophan (Figure (red) electrochemical potential in the BAM-D9 interaction area. g-h, 2D scheme of BamA-darobactin hydrogen bond interactions. Canonical β-strand hydrogen (red) bonds, side-chain interactions (blue), and distance variations when comparing DA (g) and D9 (h) were exhibited.