| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 92 === Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease produced by the pathogenic bacterium Streptococcus pyogene. SPE B is initially expressed as a 40-kDa zymogen, and subsequently converted to 28-kDa (253 residues) active protease by autocatalysis or proteolysis. Mature SPE B was shown to participate in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. Many reports suggest that SPE B serves as an important virulence factor in streptococcal infections, making it an attractive therapeutic target. In order to investigate the structure and function relationships of SPE B, we expressed SPE B and its inactive C47S mutant in E. coli and purified them to homogeneity. Since mature SPE B can be degraded by auto-proteolysis, we studied NMR structures of 28-kDa active SPE B in the presence of irreversible cysteine protease inhibitors, including L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane (E-64), L-trans-epoxysuccinly-leucylamido-(3-methyl)-butane(E-64c), iodoacetate (IAA), and iodoacetamide (IAAm). In this study we have determined the backbone 1H, 13C, and 15N resonances for the 28-kDa SPE B/E-64 complex and deduced its secondary structures from multidimensional NMR spectroscopy. Based on deuterium exchange experiment and NMR analyses, we found that 28-kDa mature SPE B/inhibitor complexes and its inactive C47S mutant have the same tertiary fold. Comparisons of the chemical shift differences between the SPE B/inhibitor complex and its C47S mutant showed that six regions, including Y15-G18, T45-A51, S135-S141, G188-F197, W212-W214, and A231-A246, are involved in the binding of inhibitors to SPE B. In addition, we found that the carboxylic acid group of the inhibitors E-64 and IAA disrupted the interactions of H of D219 and NH of T234, and H of W214 and NH of G239, resulting in the movement of the S230-N242 loop. NMR analyses reveals that the undefined S230-N242 C-terminal loop in the X-ray structure is involved in loop movement upon the inhibitor binding and may also function as a gate controlling access of the substrate to the active site. Our analyses of the chemical shift differences between the SPE B/Inhibitor complexes and C47S mutant suggest that the P2 site fits into a hydrophobic pocket formed by the V189, F197, W212, and W214 residues, and the P3 site is hydrogen bonded to the D130 and S137 residues. NMR Docking of the inhibitor E-64 into the X-ray structure of SPE B is ongoing. 3D structures of the SPE B/inhibitors complexes will be used for future rational drug design, and the resulting drugs will be applied for the clinical treatment of virulent GAS infection.
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