Investigating the Conformational Dynamics of the Outer Membrane LPS Translocon LptDE

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria that plays a crucial role in preventing toxic hydrophobic compounds from entering the cell. Newly-formed LPS molecules are extracted from the inner membrane and inserted into the outer leaflet of the OM by a multi-protein complex, comprising seven proteins: LptA-G. The heterodimer formed by the integral β-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasm to its final location on the outer leaflet of the OM. Although several crystal structures of this protein have been solved, little is known about the conformational changes following LPS binding to LptDE and the molecular details of the insertion process. Here we combine native mass spectrometry (MS) with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations (MD) to investigate the influence of substrate, lipids and inhibitor binding on the conformational dynamics of the LptDE complex. Using native MS, we captured the interaction between LptDE and its ligands and revealed preferential binding for anionic phospholipids. Moreover, we showed a non-competitive mode of action for thanatin, a recently reported inhibitor of LPS translocation. Our HDX-MS and MD data uncover LptDE segments critical for substrate and lipid binding and suggest that LPS stabilises the open state of the protein, while thanatin shifts the equilibrium towards a closed state. Furthermore, LptDE remains in the closed conformation in the presence of both LPS and thanatin, indicating that the inhibition of LPS transport played by thanatin relies on an allosteric mode of action. Our results shed light on the molecular mechanisms by which LPS is inserted into the OM and provide new insights into LPS transport inhibition, paving the way for the development of novel antimicrobial therapeutics.