Presence of substrate aids lateral gate separation in LptD.

Lipopolysaccharides (LPS) provide the outer membrane (OM) of Gram-negative bacteria with a strong protective barrier. The periplasm-spanning Lpt machinery is responsible for the transport of LPS molecules across the periplasm, culminating in insertion by the outer-membrane proteins LptD and LptE. In order to elucidate the mechanisms of LPS insertion by LptDE, we performed over 14 microseconds of equilibrium molecular dynamics simulations. Bilayer-dependent differences in the fluctuations and secondary structure of LptD's extracellular loops are observed for a pure DMPE membrane vs. a model of the OM. Furthermore, LptD's periplasmic N-terminal domain is highly dynamic, which may help to maintain the integrity of the periplasm-spanning complex amidst relative motion of the inner-membrane and outer-membrane anchored domains. In addition, our simulations demonstrate that binding of LPS substrate activates a switching between the associated and dissociated states of two lumenal loops at the interface between the β-barrel and the N-terminal domain as well as LptD's lateral gate on the microsecond timescale, neither of which is observed for the apo state. Placement of a substrate LPS molecule also causes an increase in the average separation of the LptD lateral gate strands and a lowering of the energetic barrier to lateral gate opening.