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，旨在解决的三维结构， 参与鼠疫耶尔森氏菌（鼠疫的病原体）III型分泌的蛋白质。 因为III型分泌系统（T3 SS）对于毒力是必需的，所以产生的 结构信息可以用来开发针对这种潜力的有效对策 生物恐怖分子我们已经解决了12个新的结构，并在这个过程中， 解决更多的问题，包括几种蛋白质-蛋白质复合物。在一种情况下，我们有 已经开始了基于结构的药物开发过程。细胞毒性效应物之一 耶尔森氏菌通过T3 SS注入哺乳动物细胞的蛋白质YopH是一种有效的 真核细胞样蛋白酪氨酸磷酸酶（PTP-Like protein tyrosine phosphatase，PTP-Like protein tyrosine phosphatase）。YopH使几种蛋白质去磷酸化 与真核细胞中的粘着斑相关，从而使细菌能够 避免被巨噬细胞吞噬和破坏。与Terrence Burke博士合作 Jr.（药物化学实验室，CCR）和博士罗伯特乌尔里希（USAMRIID），我们有 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结晶了其中一种，并解决了共晶体 分辨率为2.2 A。由此产生的结构信息表明， 可能提高抑制剂效力的方法，这些可能性是 目前正在探索中。此外，我们还确定了一个高分辨率的结构（1.5 A）与不可水解的六肽底物类似物复合的YopH PTB， 为我们提供了另一个开发抑制剂的起点。我们有 最近，我们扩大了结构研究的目标范围，包括毒力因子 其他潜在的生物恐怖主义因子，包括天花病毒， 土拉热弗朗西斯菌，土拉菌病的病原体。蛋白质的晶体结构 这两种来源最近都已确定。其中一个是目前的焦点， 另一个基于结构的药物开发项目。

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 A 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 A) 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.