Olsen Johan G, Dagil Robert, Niclasen Louise Meinert, Sørensen Ole E, Kragelund Birthe B
Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, Copenhagen, Denmark.
J Mol Biol. 2009 Oct 30;393(3):693-703. doi: 10.1016/j.jmb.2009.08.046. Epub 2009 Aug 25.
Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.
化脓性链球菌的侵袭性感染依赖于半胱氨酸蛋白酶——化脓性链球菌热原性外毒素B。该酶先前的结构尚未揭示其正确的活性位点构型。在此，成熟酶的晶体结构解析至1.55 Å，显示其为同型二聚体。一个亚基的丝氨酸插入另一个亚基的活性位点，为氧阴离子洞提供作用，并与活性位点半胱氨酸附近的配体配位。二聚化是成熟形式所特有的，显然是催化作用的先决条件。目前的结构支持一种三方开关系统，该系统在二聚化和底物结合时触发：（1）活性位点组氨酸从无活性构型中释放；（2）阻塞底物结合口袋的残基重新定位；（3）两个活性位点色氨酸重新定位以形成活性构型。基于目前的结构，对CA家族半胱氨酸蛋白酶的活性位点进行了扩展，并提出了脱酰基机制的详细机理。这些结果可能有助于开发针对细菌半胱氨酸蛋白酶的特异性蛋白酶抑制剂。
