Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT

Abstract

OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10 )4 M for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

“…In this context, the decrease in the a-helical structures with decreasing water content (Table 1) may be understood. Our data are in rough accordance with those of Kramer et al (2002) who found no large structural changes of OmpT secondary structure on addition of LPS. This was surprising since the protease activity of OmpT was only obtained after addition of LPS.…”

“…OmpT was recombinantly expressed in E. coli and purified from inclusion bodies as described previously (Kramer et al 2000(Kramer et al , 2002 with some modifications.…”

FtsH-dependent Processing of RNase Colicins D and E3 Means That Only the Cytotoxic Domains Are Imported into the Cytoplasm

“…1A, lane 3). Polymixin B is known to form a complex with the LPS, which are necessary for the activity of OmpT (32). The N-terminal sequence of the SC form, liberated by the purified LepB and OmpT together (Fig.…”

Structural Analysis of Oligosaccharides from Lipopolysaccharide (LPS) of Escherichia coli K12 Strain W3100 Reveals a Link between Inner and Outer Core LPS Biosynthesis

“…Experimental evidence has been obtained recently showing that certain LPS structural motifs are recognized by outer membrane proteins (Omp) and that LPS plays a role for the correct folding and activity of proteins, such as PhoE (5), OmpT (6), and FhuA (7,8). The isolation and detailed structural characterization of all different glycoforms present in LPS from Escherichia coli J-5 aided the interpretation of results from in vitro folding experiments of outer membrane proteins (5,6) and furthermore gave an insight into the biosynthesis of LPS (9). Whereas the presence and distribution of negative charges is undoubtedly very important, the highly conserved nature of carbohydrates that build the inner core region implies that they are particularly suited to fulfill certain functions.…”