Fig. 3: The open state of the periplasmic domain of ExbD is conformationally selected upon binding to TonB/HasB.

a, b 2D [¹H-¹⁵N]-TROSY spectra of ¹⁵N-labeled HasB^{Sm, peri} alone (orange) and in the presence of unlabeled ExbD^{Sm, peri} (blue). The presence of ExbD leads to the disappearance (broadening) of the peaks corresponding to the residues 46-61 of HasB^{Sm, peri} indicating binding of this region to ExbD. c Model of HasB inserted in the inner membrane: The HasB-ExbD region of interaction (orange) is located in an intrinsically disordered region (IDR) of HasB (blue) close to the inner membrane inserted N-terminal α-helix. d 2D [¹H-¹⁵N]-HMQC spectra of ¹⁵N-labeled ExbD^{Sm, peri} without (black) and with (pink) the HasB peptide (corresponding to the binding region on the HasB side). Binding of the peptide leads to signal intensity decrease and appearance of new peaks that can be assigned to the NIBS residues. e R₂ relaxation rate measurements of the free (black) and HasB peptide bound state (pink) of ¹⁵N ExbD^{Sm, peri} reveal that the binding stabilizes the open state of ExbD^{Sm, peri} characterized by the disordered nature of the NIBS residues (lower relaxation rates). This suggests a conformational selection of the open state of ExbD. Data points represent mean R₂ values obtained from fitting NMR data, with error bars reflecting the SD from Monte Carlo simulations performed for each residue, as detailed in the Methods. f Deletion of the NIBS residues of ExbDEc, peri allows for crystallization with a peptide corresponding to the binding region on the TonB side. Herein, the TonB peptide (orange) replaces the NIBS residues at the interface in between the two subunits of the ExbD dimer (pink, light pink). g Schematic mechanism: The open state of ExbD (pink, right side) is conformationally selected by TonB/HasB (blue) leading to a disorder-to-order transition of the IDR of TonB/HasB. The binding motif on the TonB/HasB side is highlighted in orange.