Tol and Ton complexes use the proton electrochemical potential to assume essential functions in Gram negative bacteria. These molecular motors are formed by the assembly of three subunits (TolA-TolQ-TolR or TonB-ExbB-ExbD) in which TolQ and ExbB are the most abundant. TolA and TonB link the TolB-Pal outer-membrane complex and the TBD transporters, respectively. Previous results indicated that TolQ-TolR and ExbB-ExbD complexes can be exchanged for the import of colicins and that TolQ-TolR can complement active transport function in absence of ExbB-ExbD.
To obtain an integrate view of the inner membrane Tol and Ton complexes, we have developed an interdisciplinary study in collaboration with different laboratories, mixing genetic, biochemical and biophysical techniques. We analyze the assembly and dynamic of the complex using cross-linking techniques in vivo. Functional studies are investigated by checking the outer membrane integrity and the cell division process for the Tol complex, and import of Iron for the Ton complex. We also use colicin and phage tools to analyze the function of the Tol and Ton systems.
Structural and functional studies on these complexes isolated from various Gram negative bacteria are investigated following five main lines in collaboration with international teams:
interactions between the complex subunits in vivo.
in vitro functional study of the complexes reconstituted in phospholipid bilayers.
structural study of soluble domain by NMR.
structural and dynamic studies of the complexes by EPR.
high-resolution structure of the membrane complexes.
The first structure/function results of such motor have been obtained with the E. coli Ton membrane complex. We have characterized the channels formed by the ExbB, ExbB/ExbD complexes and obtained the X-ray and cryo-EM high resolution structures of the ExbB/ExbD subcomplex.
The structure of the ExbB-ExbD complex.
X-ray structure Left panel: Electrostatic surface representation of ExbB showing the electropositive ‘belt’ and the electronegative ‘cap’. Blue and red shades indicate electropositivity (blue) or electronegativity (red). Central panel: The ExbB-ExbD complex highlighting the transmembrane helix of ExbD (blue) located within the transmembrane pore of the ExbB pentamer (grey). Right panel: Cryo-EM representation of the reconstructed atomic model, colored in cyan for the ExbB pentamer, red and yellow for the twoTM domains of the two ExbD subunits. For clarity only three ExbB subunits are represented. The essential Asp25 of ExbD are shown with red circles