HtrA Heat Shock Protease Interacts with Phospholipid Membranes and Undergoes Conformational Changes

Abstract

The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell survival only at temperatures above 42°C. It has been shown by genetic experiments that HtrA is an envelope protease, functioning in the periplasmic space. To clarify the cellular localization of HtrA, E. coli cells were fractionated, and HtrA was not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protein conformation released HtrA almost completely. These results, taken together with the evidence showing that HtrA functions in the periplasm, indicate that HtrA is a peripheral membrane protein, localized on the periplasmic side of the inner membrane. As the first step toward solving the problem of HtrA-membrane interactions, the structure of HtrA in the presence of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or cardiolipin (CL) was analyzed by fluorescence and Fourier-transform infrared spectroscopy. The infrared and fluorescence data indicated an interaction of HtrA with PG and CL but not with PE suspensions. Fluorescence spectroscopy revealed that this interaction was at the level of the polar head group of the phospholipid. In the PG/HtrA system, small changes were observed in the HtrA secondary structure and a remarkable decrease of the thermal stability of the protein, which suggested changes in HtrA tertiary structure. This suggestion was supported by fluorescence data that showed a shift of the fluorescence emission spectrum of HtrA tyrosine residues in the presence of PG and a reduced fluorescence intensity, phenomena not observed in the presence of PE or CL suspensions. Infrared data revealed also that the interaction of HtrA with PG leads to a protection of unfolded protein against aggregation at relatively low temperatures. The conformational changes of HtrA in the presence of PG influenced the proteolytic activity of HtrA by increasing it at the temperatures 37-45°C and inhibiting it at 50-55°C. CL inhibited HtrA activity at all of the temperatures tested.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

“…Proteoliposomes that contain synthetic lipids may also have practical utility for experiments in which it is desirable or necessary to use bilayers that have a specific lipid composition to study the assembly of an OMP of interest. Furthermore, the ability to choose the membrane environment should facilitate examination of the hypothesis that the function of periplasmic chaperones such as Skp and DegP requires interactions with specific lipid head groups (36,70).…”

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

“…HefA is homologous to TolC, an outer membrane protein from E. coli (37,38), while HP1564 is homologous to an outer membrane protein of Pasteurella hemolytica (39). In contrast, the ABC transporter of iron, CeuE, is likely to be localized in the periplasm based on data from E. coli (40). However, a subpopulation of this protein might still be surface-exposed like other classic periplasmic proteins that have been found in this and previous studies to be surface-exposed in H. pylori (HtrA, ␥-glutamyltranspeptidase).…”

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

“…It is a membranebound metalloprotease, where the proteolytic and ATPase domains also reside in the same polypeptide. DegP is a peripheral membrane Ser protease located in the periplasmic side of the cytoplasmic membrane in E. coli (Skorko-Glonek et al, 1997).Analysis of prokaryotic and eukaryotic genomes reveals that the number of genes encoding the aforementioned proteases has increased during evolution. For instance, the E. coli genome contains single genes encoding Lon, FtsH, ClpP, A and X, and three DegPlike encoding genes.…”

“…It is a membranebound metalloprotease, where the proteolytic and ATPase domains also reside in the same polypeptide. DegP is a peripheral membrane Ser protease located in the periplasmic side of the cytoplasmic membrane in E. coli (Skorko-Glonek et al, 1997).…”