Recombinant Escherichia coli Rhomboid protease glpG (glpG)

1. we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). PMID: 28065266
2. The rhomboid protease GlpG, was found to promote extraintestinal pathogenic Escherichia coli survival within the mouse intestinal tract in the presence of the intact natural microbiota. PMID: 28373355
3. Unbinding of TM5 from the rest of the structure exposes GlpG's active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site. PMID: 26858402
4. Studied high-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid; this provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. PMID: 25307614
5. performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2-3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli PMID: 26056273
6. Peptidyl-chloromethylketones (CMKs) derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. PMID: 25216680
7. Structure and mechanism of rhomboid protease. PMID: 23585569
8. Data indicate that the rhomboid GlpG (ecGlpG) cytoplasmic domain exists as a dimer with extensive domain swapping between the two monomers. PMID: 23353827
9. GlpG is a rhomboid membrane protease which recognizes features of transmembrane regions of substrates. Cleavage occurs between Ser & Asp in a region of high local hydrophilicity, which might be located juxtamembrane rather than intramembrane position PMID: 16216077
10. 2.1 A resolution crystal structure of the GlpG core domain PMID: 17051161
11. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix alpha5 bent away from the rest of the molecule PMID: 17099694
12. the crystal structure of GlpG in a more open conformation, where the capping loop L5 has been lifted, exposing the previously buried and catalytically essential Ser-201 to outside aqueous solution PMID: 17277078
13. The functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. PMID: 17463085
14. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment. PMID: 17493126
15. L1 loop is partially embedded in the membrane, and showed that alanine substitution of a highly preferred tryptophan (Trp136) at the distal tip of the L1 loop near the lipid:water interface reduced GlpG proteolytic activity. PMID: 17976648

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KEGG: ecj:JW5687

STRING: 316385.ECDH10B_3598