Citation

Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix. LptC directly binds LPS and is hypothesized to be involved in the transfer of LPS to another periplasmic protein, LptA. Purified and in solution, LptC forms a dimer. Here, point mutations designed to disrupt formation of the dimer are characterized using site-directed spin labeling double electron electron resonance (DEER) spectroscopy, light scattering, circular dichroism, and computational modeling. The computational studies reveal the molecular interactions that drive dimerization of LptC and elucidate how the disruptive mutations change this interaction, while the DEER and light scattering studies identify which mutants disrupt the dimer. And, using electron paramagnetic resonance spectroscopy and comparing the results to the previous quantitative characterization of the interactions between dimeric LptC and LPS and LptA, the functional consequences of monomeric LptC were also determined. These results indicate that disruption of the dimer does not affect LPS or LptA binding and that monomeric LptC binds LPS and LptA at levels similar to dimeric LptC. This article is protected by copyright. All rights reserved. 2018 The Protein Society.