LPS Binding to LptA

Abstract

Gram-negative bacteria, such as Escherichia coli, have an asymmetric outer membrane (OM) with an outer leaflet rich in lipopolysaccharide (LPS). LPS is a large lipid essential for survival in Gram-negative bacteria and a major factor in the protection of bacteria from adverse environmental stresses. LPS is transported across the inner membrane by MsbA and through the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) proteins LptA, LptC, LptDE, and LptFGB2, the majority of which are essential to Gram-negative bacterial survival. LptA is a periplasmic protein that has been shown to form concentration dependent oligomers that stack end-to-end. The LptA protein fold is speculated to bind the LPS acyl chains in the central pocket of the protein, to protect the hydrophobic region as the molecule crosses the soluble periplasm. In the studies presented here, the binding of LPS at individual sites throughout the LptA protein is characterized using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy. To determine the location of LPS binding on LptA, fifteen positions located in and around the hypothesized LPS binding pocket on LptA were mutated to cysteine, purified, spin labeled at the unique introduced cysteine, and tested for mobility changes due to the presence of excess exogenous LPS. Our data suggest a 1:1 ratio for the LPS:LptA complex and an affinity gradient from one end of the protein to the other based on Bmax and Kd values quantitatively determined by EPR spectroscopy.