Streptopain (又稱為Streptococcal pyrogenic exotoxin B，簡稱 SPE B)，它是由具致病力的化膿性鏈球菌 (Streptococcus pyogenes) 所表現出來的一種細胞外毒素。SPE B 是一種 cysteine protease，一開始是以酶原態 42-kDa 的形式被表現出來，緊接著會經由自動催化或經由其他蛋白酶的活化而轉成活化態 28-kDa 的形式。許多文獻已指出 SPE B 是此致病菌的一個重要毒性因子，其在病菌感染過程中會藉由切割宿主蛋白進而促使病菌的散佈、入侵、加重發炎反應並抑制傷口癒合等，也指出 SPE B 之所以對細胞或組織有如此大的傷害完全在於其本身的酵素催化特性。為了瞭解 SPE B 為何會有如此廣泛的受質專一性及往後能設計出針對 SPE B 的藥物，我們利用 NMR 分析其動力學特性並定出其 3D 結構以進一步了解其功能。利用加入不同抑制劑 (general inhibitor) 去模擬 SPE B 的受質，發現 SPE B 在結合上抑制劑後有六個區域有明顯化學位移變化 (chemical shift perturbation)，對照到 X-ray 所解出的 42-kDa 酶原態結構後，其中五個區域位於結構上的催化中心，然而最特別的是結構中所沒解出來的 C 端圈環竟然也有很明顯的化學環境變化，因此我們認為C端圈環對於蛋白與受質的結合或催化過程或許扮演了重要的角色。蛋白質的內部運動與其功能息息相關，利用 NMR 分析 SPE B 的動力學發現到 SPE B 整個蛋白內部都是很固定的 (rigid)，唯獨 C 端與催化圈環具有高度活動性 (flexible)，此外當 SPE B 與抑制劑結合後 C 端及催化圈環活動性會增高，若抑制劑帶有一個負電荷的羧基 (carboxyl group)如E-64、E-64c與IAA，又會更提升此二個圈環活動性。從NMR圖譜分析中發現到 C 端與催化圈環彼此間有相互作用，而這樣的相互作用會因為蛋白結合上抑制劑後而消失。我們也解出了SPE B/E-64 複合蛋白與突變蛋白 C47S 的 28-kDa 活化態結構，比較二者結構發現到此二個圈環在 C47S 中相距較近 (5.2 Å)，而在 SPE B/E-64 複合蛋白中相距較遠 (11.7 Å)。從功能性分析中也發現到若將催化圈環旁的 V189 及 C 端圈環上的 G239 進行單點突變將會使 SPE B 的催化活性分別降低達 10 倍與 60 倍之多。綜合以上所述，C 端與催化圈環在酵素催化過程及受質結合與產物釋放中或許扮演了重要的角色。除此之外，G136 loop 與 W212-W214 loop 在木瓜酵素家族中是保留的結構，此二個區域的突變亦會大大降低酵素活性，從動力學結果中發現到此二個 loops 具有很高的 Rex 數值，且在圖譜中發現到 G136 loops 與抑制劑 E-64 有相互作用，將圖譜分析結果利用 docking 的方式模擬出 SPE B/E-64 複合蛋白的結構。此研究不僅更了解 Streptopain 的內部催化過程，特別是催化圈環及 C 端圈環在受質結合及催化過程中所扮演的角色，在針對 Streptopain 的專一性藥物設計上亦提供了重要的訊息。

Streptopain (streptococcal pyrogenic exotoxin B; SPE B) is an extracellular cysteine protease expressed by the pathogenic bacterium Streptococcus pyogenes. SPE B is initially expressed as a 42-kDa zymogen and subsequently converted to a 28-kDa active protease by autocatalysis or proteolysis. Many reports have shown that SPE B is an important virulence factor in streptococcal infection such as the dissemination, colonalization, invasion of bacteria, and inhibition of wound healing. To understand why SPE B has broad substrate specificity and to design the drugs for SPE B, we used NMR spectroscopy to determine the 3D structure and dynamics of the SPE B/inhibitor complex. Comparisons of NMR chemical shift differences between the SPE B/inhibitor complexes and the C47S mutant showed that six regions, including Y15-G18, T45-A51, S135-S141, G188-F197, W212-W214, and A231-A246, were involved in the binding of inhibitors to SPE B. The result suggests that the A231-A246 loop, which is unobserved in the crystal structure, may play important roles in substrate binding and recognition. Dynamics analysis of the SPE B/E-64, SPE B/E-64c, and SPE B/IAA complexes showed that the catalytic (G188-F197) and C-terminal (A231-G240) loops were the most flexible regions with motions on the s/ms and ps/ns timescales. In contrast to the complexes with inhibitors containing carboxylic acid moiety, these loops of the C47S mutant and the SPE B/IAAm complex were less flexible. 3D structures of C47S mutant and SPE B/E-64 complex were determined by NMR spectroscopy. The distances between C-terminal and the catalytic loops of the C47S mutant and the SPE B/E-64 complex were 5.94 and 11.62 Å, respectively. This is consistent with NOE analysis that interactions between residues in H195 and A231, and in V192 and A238 were observed in the C47S mutant. Our mutagenesis study also showed that mutations on V189, the residue of the catalytic loop, and on G239, the residue of the C-terminal loop, caused an 11- to 61-fold decrease in activity, suggesting that they were important for the substrate binding. In this study, we found that not only the catalytic loop but also the C-terminal loop play and an important role in the substrate binding and enzyme catalysis of SPE B. We also found that the S135-S141 and W212-W214 loops, the conserved regions in the papain superfamily, have conformational exchange with high Rex values. Based on 15N/13C-edited and -filtered experiments, the NOE interactions indicated that G136 of the S135-S141 loop interacts with the inhibitor, E-64. The conformation of E-64 will be docked into 3D structure of SPE B with the observed intermolecular NOEs. These finding indicates that the C-terminal and the catalytic loops of SPE B play an important role in its substrate binding, and the results will facilitate rational drug design of SPE B.

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