熱原性鏈球菌外毒素B (Streptococcal pyrogenic exotoxin B 簡稱SPE B)，它是由化膿性鏈球菌(Streptococcus pyogenes)所產生的一種細胞外毒素，根據催化區分析將它歸屬於半光胺酸蛋白?的一種。SPE B會經過自動催化(autocatalysis)或蛋白?分解(proteolysis)作用，將40-kDa的?原(zymogen form SPE B簡稱zSPE B或ProSPE B)去掉118個胺基酸的propeptide變成28-kDa活化態的蛋白?(簡稱mSPE B)。SPE B在In vitro下可以分解細胞間質的主成份fibronectin及vitronectin、作用於interleukin-1β (IL-1β) precursor並產生活化型的IL-1β而加重發炎反應、誘導吞噬細胞進行apoptosis及降低其吞噬能力等，故SPE B被認為是化膿性鏈球菌的重要毒性因子而成為極具吸引力的治療標的。為瞭解SPE B結構與功能間的相互關係，我們將SPE B和其突變蛋白分別送入大腸桿菌系統誘發蛋白表現，經過Ni+2-chelate affinity 管柱純化。SPE B在純化過程中會自動轉換成28-kDa的活化態形式，所以在純化的過程我們加入一些半光胺酸蛋白?不可逆的抑制劑去抑制SPE B的降解，這些抑制劑包含E-64，E-64c，IAA與IAAm這四者。在我們的研究中，我們利用同位素標定蛋白(2H,13C,15N triple- and 13C,15N double-labeled samples)完成了96%骨架的判定; 此外利用D2O交換的實驗，推測野生株的SPE B與突變株C47S本身的二級、三級構造類似。進一步比較SPE B/抑制劑複合蛋白與C47S突變蛋白的化學位移的差異，我們發現有六區有可能與抑制劑結合有關，分別是Y15-G18, T45-A51, S135-S141, G188-F197, W212-W214, and A231-A246。比較X-ray所解出的42-kDa的C47S，我們發現X-ray沒有看見的C-terminal loop(S230-N242)，本身在抑制劑結合後會發生移動的現象，這似乎暗示著C-terminal loop本身可以當做一個閘門去調節基質進入活性區域內。利用NMR的分析，我們使用的抑制劑，如E-64與IAA，本身帶有羧基，帶負電，它會去打斷D219 NH 和T234 NH , 與W214 H 和 G239 NH的作用，造成loop(S230~N242)的移動。根據SPE B/抑制劑複合蛋白之間的化學位移差異，我們假設E-64有可能結合到SPE B的位置。其中E64中的P2 ，它會與SPE B上V189，F197，W212與W214所形成的疏水性區域有凡得瓦爾力作用; 此外E64的P3會與SPE B的D130 與S137有氫鍵作用。目前仍在進行NMR docking來計算我們的結構。本研究的結果將可做為設計屬於SPE B專一性藥物 (rational drug design)的基礎。

　　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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