|The catalytic mechanism. A, GlpG catalyzes the hydrolysis of DCI to form an α-hydroxy acid. The complex between 7-amino-4-chloro-3-methoxy isocoumarin and GlpG is stabilized by two covalent bonds. B, The covalent adduct between DFP and GlpG mimics the tetrahedral transition state. C and D, The crystal structures of GlpG in complex with isocoumarin and DFP, respectively (Protein Data Bank codes 2XOW and 3TXT; ref. 25, 33). E, A hypothetical model of substrate (green) bound to rhomboid protease (side view; 90° from that in Fig. 1B). The protease’s TM helices are shown as cylinders, and the loops are omitted for clarity. The substrate’s extended cleavage site and helical TM segment are connected by a sharp turn (green dots). According to this model, Ala-253 is adjacent to the side chain of substrate’s P1 residue (insert). The red arrows indicate the scissile bond. Click on the image to see a larger version of it.
Rhomboid proteases are a family of enzymes, each with a common catalytic core made up of six transmembrane segments, that cleave membrane-protein substrates near the amino-terminus of the transmembrane domain. They were first identified from a genetic screen in Drosophila, where flies lacking this protein would express a pointy head skeleton phenotype. Homologs to the rhomboid protease from Drosophila have been discovered in many prokaryotes and other eukaryotes, and they are involved with a wide variety of biological functions. Rhomboid proteases are also members of a distinct class of proteases called intramembrane-cleaving proteases, or I-CLiPs, a term that emphasizes their ability to operate within the hydrophobic region of the lipid bilayer. E. coli rhomboid protease GlpG was the first I-CLiP to have its crystal structure solved; however, it remains unclear how it functions within the membrane.
In a minireview recently published in The Journal of Biological Chemistry, Ya Ha and Yi Xue of the Yale School of Medicine and Yoshinori Akiyama of Kyoto University discuss work done to determine the mechanism of rhomboid proteases. The minireview specifically focuses on research done on the catalytic mechanism and conformation changes in the catalytic core of the E. coli rhomboid protease GlpG.
One of the experiments reviewed showed that a serine residue from the catalytic center of GlpG is bonded covalently to a mechanism-based inhibitor, indicating that this protease may function via a classical mechanism. Other studies reviewed put forward a top-down model, suggesting that rhomboid proteases may cleave peptide bonds initially buried in transmembrane regions as well as those outside the transmembrane domains. One of the studies also identified a conserved motif specifically recognized by rhomboid proteases, suggesting that rhomboid proteases use a common and specific mechanism to recognize their substrates. However, not all rhomboid substrates share this motif, indicating that other specificity-determining mechanisms exist.
The authors of the minireview propose that further research should focus on interactions between rhomboid proteases and the lipid bilayer, generating additional crystal structures where they are in complex with peptide substrate analogs, and should examine their role in the life cycle on medically relevant parasites such as T. gondii and P. falciparum.
The authors write, “The biological functions of many related rhomboid proteins are now known, and there is optimism that the pace of such discoveries will only quicken in the near future. The crystal structures of E.coli and H. influenza GlpGs have provided a framework for in-depth probing of the membrane protein’s mechanism of action.”
Anna Shipman (email@example.com) is a Ph.D. student in the School of Biological Sciences at the University of Missouri-Kansas City.