(A) Cartoon of the E. coli cell envelope, with IgaA in the IM and RcsF and OmpA in the OM. RcsF, an OM lipoprotein, interacts with OmpA. When in complex with RcsF, OmpA adopts its two-domain conformation, with an N-terminal β-barrel embedded in the OM and a C-terminal domain soluble in the periplasm. The periplasmic domain of OmpA interacts with RcsF, likely competing with IgaA for RcsF binding. Copy numbers are from Li et al., 2014. (B) Impact of over-expressing IgaA on the OmpA-RcsF complex (left). Cells harboring pSC231, an empty vector used as control, or pIgaA (pSC237, expressing IgaA-Flag3 from an IPTG-inducible promoter) were harvested at mid-log phase and incubated without (lanes 1–6) or with (lanes 7–12) BS3. Protein samples were immunoblotted with α-Flag (upper panel), α-RcsF (middle panel), or α-OmpA171-325 (lower panel) antibodies. IgaA-Flag3 was expressed in WT (lanes 2–4 and 8–10), ∆rcsF (lanes 5 and 11), and ∆ompA cells (lanes 6 and 12) with the indicated IPTG concentrations. Quantitation (see Figure 5—source data 1 for details) of the IgaA-RcsF and OmpA-RcsF complexes detected by the anti-RcsF antibodies is shown (right panel).* and **, non-specific bands detected by the polyclonal α-RcsF antibody. (C) Plot of the chemical shift perturbation (CSP) measured on OmpA resonances as a function of the RcsF:OmpA ratio. Only the residues with significant CSP (colored in blue in Figure 4B) are plotted and used to fit the KD. (D) The interaction between RcsF and the periplasmic domain of IgaA was probed by biolayer interferometry (BLI). Sensortips carrying immobilized RcsF were dipped into increasing concentrations of IgaA (5.9, 8.9, 13.3, 20, 30 nM) from 0 to 1000s then into buffer (1000–2000s). Association and dissociation phases were fitted (red lines) to extract a KD value. Residuals from the fits are shown at the bottom of the panel (see Figure 5—source data 2 for statistics).