Fig. 6: TAM-catalyzed OMP assembly requires an interaction between TamA and the β signal of the substrate.

a The amino acid sequences of E. coli K-12 TamA and BamA β strands 1 and 16 (β1 and β16) and extracellular loop 6 (eL6) aligned based on the crystal structures of TamA (PDB: 4C00⁴²) and BamA (PDB: 5D0O²⁴). The BamA motifs conserved in Proteobacteria are indicated by asterisks⁸⁶. b The binding of TamA(β1) to EspP(β12) (right) was predicted based on the BamA(β1)-EspP(β12) structural model (PDB: 7TTC³⁴) (left). Seven H-bonds were predicted between TamA(β1) and EspP(β12) (right) and are denoted by solid red lines. Two H-bonds are predicted between TamA residues Tyr²⁷⁴ and Asn²⁶⁶ and EspP [TamA(Y274)-EspP(A1291) and TamA(Y274)-EspP(V1292); TamA(N266)-EspP(C1299) and TamA(N266)-EspP(F1300)], but only the closer H-bonds are shown. c Urea denatured wild-type (WT) OmpA or EspPΔ5’, β signal mutants (β) (OmpA^{Y189A, F191A} or EspPΔ5’^{Y1298A, F1300A}), or folding deficient controls (C) (OmpA^{L98R, V100R} or EspPΔ5’^I1119R) were incubated with 2 μM BAM/PLE or TAM/PLE proteoliposomes at 30 °C for 2 min (for OmpA) or 15 min (for EspPΔ5’). Unheated OmpA or heated EspPΔ5’ samples were subjected to SDS-PAGE, and assembly was monitored by Western blot using the appropriate anti-C terminal peptide antiserum. A representative experiment is shown. Three independent experiments were performed, and the other two are shown in Supplementary Fig. 10a. d The binding of darobactin to TamA(β1) (right) was predicted based on the structure of darobactin-bound BAM (PDG: 7NRI) (left)⁹¹. The seven H-bonds predicted between darobactin and TamA(β1) are denoted by solid red lines. e 2 μM BAM or TAM proteoliposomes were incubated with darobactin at 30 °C for 5 min. The amount of darobactin added to the reaction was varied to achieve the indicated darobactin-to-proteoliposome (PL) ratios. Urea-denatured OmpA or EspPΔ5’ was added to the reaction and incubated at 30 °C for 15 min. Unheated OmpA or heated EspPΔ5’ samples were subjected to SDS-PAGE, and assembly was monitored by Western blot using the appropriate anti-C terminal peptide antiserum. A representative experiment is shown. Three independent experiments were performed and a statistical analysis is shown in Supplementary Fig. 10b.