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Topology of the outer membrane phospholipase A of Salmonella typhimurium
K B Merck1, H de Cock, H M Verheij
1Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, The Netherlands. k.b.merck@ato.dlo.nl
Abstract:
The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.
Insights
This study investigated the structure of outer membrane phospholipase A (OMPLA) in Salmonella typhimurium using epitope insertion. Results largely support the proposed 14-transmembrane beta-strand model, identifying surface-exposed loops and key catalytic residues.
Area of Science:
- Microbiology
- Structural Biology
- Protein Topology
Background:
- The outer membrane phospholipase A (OMPLA) in Enterobacteriaceae is hypothesized to have a specific membrane-spanning structure.
- Understanding OMPLA topology is crucial for elucidating its function and interactions within the bacterial outer membrane.
Purpose of the Study:
- To experimentally determine the topology of outer membrane phospholipase A (OMPLA) from Salmonella typhimurium.
- To validate the proposed model of OMPLA spanning the membrane via antiparallel beta-strands and identify surface-exposed loops.
Main Methods:
- Utilized the epitope insertion method by introducing unique BamHI sites into the pldA gene.
- Inserted linkers encoding a 16-amino-acid B-cell epitope at various positions.
- Assessed protein assembly via heat modifiability and epitope accessibility using immunofluorescence and trypsin accessibility assays.
Main Results:
- Immunofluorescence confirmed cell surface exposure for three of seven predicted loops.
- Trypsin accessibility experiments verified two additional loops as cell surface exposed and supported periplasmic localization for three turns.
- Results largely support the proposed 14-transmembrane beta-strand topology model for OMPLA.
- Specific mutations (Glu66Pro, Glu247Gly) significantly reduced OMPLA enzymatic activity, suggesting critical roles in catalysis.
Conclusions:
- The study provides strong experimental evidence supporting the proposed topology model of OMPLA.
- Identified specific residues essential for OMPLA catalytic activity, offering insights into its enzymatic mechanism.