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.
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