Crystal Structure of the Yersinia Protein-tyrosine Phosphatase YopH Complexed with a Specific Small Molecule Inhibitor

Abstract

The pathogenic bacteria Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to bubonic plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a K i value of 25 M for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-Å resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors. Protein tyrosine phosphatases (PTPs)1 are involved in the regulation of numerous cell functions including growth, differentiation, motility, cell-cell interactions, metabolism, gene transcription, and the immune response (1, 2). In vivo, tyrosine phosphorylation is a reversible and dynamic process. The phosphorylation states of proteins are governed by the opposing actions of protein tyrosine kinases, which catalyze protein tyrosine phosphorylation, and PTPs, which are responsible for dephosphorylation. Hundreds of protein kinases and protein phosphatases and their substrates are integrated within an elaborate signal-transducing network. The defective or inappropriate operation of this network is at the root of such widespread diseases including cancers and diabetes.

Activity-based probes for protein tyrosine phosphatases

“…The ability of BBP-based probes to target the PTP active site was expected because BBP mimics pTyr, which is known to occupy the PTP active site. Indeed, the K I values for probe I and BBP are similar to competitive inhibition constants measured for benzylphosphonate, a nonhydrolyzable pTyr mimetic (11). Further evidence in support of the inactivation's being directed to the active site included the fact that arsenate, a competitive PTP inhibitor, was able to protect PTP from BBP-mediated inactivation.…”

“…The inactivation reaction was initiated by the addition of a 5-l aliquot of PTP stock to a 45-l solution containing appropriately diluted probe I (final concentration of DMSO, 5%). At appropriate time intervals, aliquots of 2 l were removed from the reaction and added to a 200-l solution containing 20 mM pnitrophenyl phosphate (pNPP) in pH 6.0 buffer at 30°C (11). The kinetic parameters of the inactivation reaction were obtained by fitting the data to the following equations: Western Blot Analyses of the Labeling Reaction.…”

“…The PTPs share a conserved active site that recognizes aryl phosphates and a common catalytic mechanism that use a highly reactive nucleophilic Cys residue (6). This Cys (Cys-403 in YopH) displays an unusually low pK a of Ϸ5 (22) and is situated at the bottom of the pTyr-binding pocket such that its S␥ atom is poised 3 Å from the phosphorus atom of pTyr (11). In the catalytic mechanism, the active site Cys initiates a nucleophilic attack on the phosphorus, leading to the formation of a thiophosphoryl enzyme intermediate.…”

Aryl Vinyl Sulfonates and Sulfones as Active Site-Directed and Mechanism-Based Probes for Protein Tyrosine Phosphatases

“…17 Other recombinant PTPs were expressed and purified as described previously. [18][19][20] Non-PTP enzymes were purchased from Sigma-Aldrich and stored at −20 °C.…”

The Sulfogalactose Moiety of Sulfoglycosphingolipids Serves as a Mimic of Tyrosine Phosphate in Many Recognition Processes

“…Furthermore, the co-crystal structure of a Yersinia tyrosine phosphatase (YopH) complexed with a small molecule inhibitor has been solved (52). This inhibitor is p-nitrocatachol sulfate, the same compound that was co-crystallized in the substrate binding site of arylsulfatase (Fig.…”