Structural Proteomics of the Yersinia Yop Virulon

我们在生产大量结晶级蛋白质方面的成功导致了一个小规模的结构基因组学项目，旨在解决鼠疫耶尔森氏菌（鼠疫的病原体）中参与III型分泌的蛋白质的三维结构。由于III型分泌系统（T3 SS）是必不可少的毒力，由此产生的结构信息可以用来制定有效的对策，这种潜在的生物恐怖主义的代理。我们已经解决了12个新的结构，并正在解决更多的问题，包括几个蛋白质-蛋白质复合物。在一种情况下，我们已经开始了基于结构的药物开发过程。耶尔森氏菌通过T3 SS注入哺乳动物细胞的细胞毒性效应蛋白之一YopH是一种有效的真核生物样蛋白酪氨酸磷酸酶（PTP 3）。YopH使真核细胞中与粘着斑相关的几种蛋白质去磷酸化，从而使细菌能够避免巨噬细胞的吞噬和破坏。与Terrence Burke Jr博士合作。（药物化学实验室，CCR）和Robert Ulrich博士（USAMRIID），我们已经鉴定了几种抑制YopH的化合物，其IC 50值在低微摩尔范围内。到目前为止，我们已经成功地用酶使其中之一结晶，并以2.2的分辨率解决了共晶结构。由此产生的结构信息提出了几种可能提高抑制剂效力的方法，目前正在探索这些可能性。此外，我们已经确定了YopH PTR与不可水解的六肽底物类似物复合物的高分辨率结构（1.5），为我们提供了开发抑制剂的另一个起点。我们最近扩大了结构研究的目标范围，包括来自其他生物恐怖主义潜在因子的毒力因子，包括天花病毒和土拉菌病病原体土拉弗朗西斯菌。最近已经确定了这两种来源的蛋白质的晶体结构。其中之一目前是另一个基于结构的药物开发项目的重点。

Our success in producing large quantities of crystallization-grade proteins led to a small-scale structural genomics project aiming to solve the three-dimensional structures of proteins involved in Type III secretion in Yersinia pestis, the causative agent of plague. Because the Type III secretion system (T3SS) is essential for virulence, the resulting structural information could be used to develop effective countermeasures for this potential agent of bioterrorism. We have already solved 12 novel structures and are in the process of solving more of them, including several protein-protein complexes. In one case, we have already begun the process of structure-based drug development. One of the cytotoxic effector proteins that Yersinia injects into mammalian cells via the T3SS, YopH, is a potent eukaryotic-like protein tyrosine phosphatase (PTPase). YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells, thereby enabling the bacterium to avoid phagocytosis and destruction by macrophages. In collaboration with Dr. Terrence Burke Jr. (Laboratory of Medicinal Chemistry, CCR) and Dr. Robert Ulrich (USAMRIID), we have identified several compounds that inhibit YopH with IC50 values in the low micromolar range. Thus far we have managed to crystallize one of these with the enzyme and solve the co-crystal structure at 2.2 resolution. The resulting structural information suggested several ways in which the potency of the inhibitor might be improved, and these possibilities are currently being explored. In addition, we have determined a high-resolution structure (1.5 ) of the YopH PTPase in complex with a nonhydrolyzable hexapeptide substrate analog, providing us with yet another starting point for the development of inhibitors. We have recently expanded our range of targets for structural studies to include virulence factors from other potential agents of bioterrorism, including the variola major (smallpox) virus and Francisella tularensis, the causative agent of tularemia. Crystal structures of proteins from both of these sources have recently been determined. One of them is currently the focus of another structure-based drug development project.