Articles High-Resolution Structure of the Yersinia pestis Protein Tyrosine Phosphatase YopH in Complex with a Phosphotyrosyl Mimetic-Containing Hexapeptide ‡ Jason Phan, # Kyeong Lee, § Scott Cherry, # Joseph E. Tropea, # Terrence R. Burke, Jr., § and David S. Waugh* ,# Macromolecular Crystallography Laboratory and Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702 ReceiVed June 19, 2003 ABSTRACT: Yersinia pestis, the causative agent of bubonic plague, secretes a eukaryotic-like protein tyrosine phosphatase (PTPase) termed Yersinia outer protein H (YopH) that is essential for virulence. We have determined, for the first time, the crystal structure of the YopH PTPase domain in complex with a nonhydrolyzable substrate analogue, the hexapeptide mimetic Ac-DADE-F 2 Pmp-L-NH 2 . As anticipated, the mode of ligand binding in the active site is similar to the way in which the corresponding phosphohexapeptide binds to the structurally homologous human PTP1B. Unexpectedly, however, the crystal structure also revealed a second substrate-binding site in YopH that is not present in PTP1B. The mode of binding and structural conformation of the hexapeptide analogue is quite different in the two sites. Although the biological function of the second substrate-binding site remains to be investigated, the structure of a substrate analogue in the active site of Y. pestis YopH opens the door for the structure- based design and optimization of therapeutic countermeasures to combat this potential agent of bioterrorism. Yersinia pestis utilizes a contact-dependent (type III) secretion apparatus to inject six cytotoxic effector proteins (YopE, YopH, YopM, YopJ/P, YopT, and YpkA) directly into the cytosol of mammalian cells, where they conspire to defeat the innate immune response of the infected organism by interfering with signaling pathways that regulate cyto- skeletal dynamics and inflammation (1-3). One of the effec- tors, YopH, is a powerful eukaryotic-like protein tyrosine phosphatase (PTPase). Upon translocation into mammalian cells, YopH disrupts signal transduction pathways required for the attachment of cells to the extracellular matrix and phagocytosis by dephosphorylating a variety of proteins associated with the focal adhesion, including focal adhesion kinase (p125 FAK ), paxillin, p130 cas , p105 casL , Fyn-binding protein, and the Src-associated adaptor protein SKAP-HOM (4-9). Because its PTPase activity is essential for virulence (10), YopH is a valid molecular target for antiplague therapeutics that could be used to combat weaponized strains that have been engineered to be resistant to conventional antibiotics. YopH is a modular protein that is composed of two inde- pendently folded domains. The N-terminal domain (residues 1-130) binds tyrosine-phosphorylated proteins in a phos- phoryl-dependent manner and evidently plays a role in the recognition of at least some substrates in vivo (5, 11). The structure of the N-terminal domain of YopH (12, 13) does not resemble those of the eukaryotic Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains, nor does it bind phosphotyrosine in a similar manner (14). The C-terminal domain of YopH (residues 164-468) is the seat of its PTPase activity (15). The structure of the Yersinia YopH PTPase domain is very similar to the structures of eukaryotic PTPases (16), suggesting that it may have been acquired by lateral gene transfer. The X-ray crystal structure of the catalytic domain of YopH was among the first PTPase structures to be solved (17). Phosphate-, tungstate-, nitrate-, and vanadate-bound structures were subsequently determined (17-20). Although these structures provided valuable information about enzyme- anion interactions and the reaction mechanism, they did not reveal how YopH recognizes its phosphotyrosyl substrates. Such information is required for the structure-based design of more potent and specific inhibitors of the Yersinia PTPase. In this report, we describe the structure of the catalytic domain of YopH in complex with a hexapeptide mimetic (Ac-DADE-F 2 Pmp-L-NH 2 ) derived from an autophospho- rylation site of the epidermal growth factor receptor cyto- plasmic domain. MATERIALS AND METHODS Synthesis of the Phosphotyrosyl Mimetic-Containing Hexapeptide. The nonhydrolyzable substrate analogue Ac- DADE-F 2 Pmp-L-NH 2 was first reported as a high affinity inhibitor of PTP1B (21). The peptide employed in the present ‡ Coordinates and structure factors have been deposited in the RCSB protein databank (http://www.rcsb.org) under the accession code 1QZ0. * Corresponding author: e-mail: waughd@ncifcrf.gov; fax: (301) 738-6255. # Macromolecular Crystallography Laboratory. § Laboratory of Medicinal Chemistry. 13113 Biochemistry 2003, 42, 13113-13121 10.1021/bi030156m This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 10/25/2003