Structural and Functional Characterization of the Histidine Phosphatase Domains of Human Sts-1 and Sts-2

Abstract

The suppressor of T-cell signaling (Sts) proteins, Sts-1 and Sts-2, are homologous phosphatases that negatively regulate signaling pathways downstream of the T-cell receptor. Functional inactivation of Sts-1 and Sts-2 in a murine model leads to resistance to systemic infection by the opportunistic pathogen, C. albicans. This suggests that modulation of the host immune response by inhibiting Sts function may be a viable strategy to treat these deadly fungal pathogen infections. To better understand the molecular determinants of function and structure, we characterized the structure and steady-state kinetics of the histidine phosphatase domains of human Sts-1 (Sts-1HP) and Sts-2 (Sts-2HP). We solved the X-ray crystal structures of Sts-1HP, unliganded and in complex with sulfate to 2.5 Å and 1.9 Å, respectively, and the structure of Sts-2HP with sulfate to 2.4 Å. The steady-state kinetic analysis shows, as expected, that Sts-1HP has a significantly higher phosphatase activity than that of Sts-2HP, and that the human and mouse proteins behave similarly. In addition, comparison of the phosphatase activity of full-length Sts-1 protein to Sts-1HP reveals similar kinetics, indicating that Sts-1HP is a functional surrogate for the native protein. We also tested known phosphatase inhibitors and identified that the SHP-1 inhibitor, PHPS1, is a potent inhibitor of Sts-1 (Ki of 1.05 ± 0.15 μM). Finally, we demonstrated that human Sts-1 has robust phosphatase activity against the substrate, Zap-70, in a cell-based assay. Collectively, these data suggest that the human Sts proteins are druggable targets and provides a structural basis for future drug development efforts.

The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges

“…These characteristics include pH-buffering, hydrogen bonding, aromaticity, coordination bonds with transitional metals, and alkylation of the ring, which modifies the hydrophobicity of the imidazole ring. Various biological systems have utilized these properties of histidine and its imidazole ring to sustain life such as enzymatic activity [ 1 ], transcriptional factors [ 2 ], the release of oxygen from hemoglobin [ 3 ], and antimicrobial activity [ 4 ]. Investigators have adopted these properties of histidines/imidazole to develop improved carriers of nucleic acids.…”

Discovery and Characterization of Two Classes of Selective Inhibitors of the Suppressor of the TCR Signaling Family of Proteins

“…Drug discovery efforts targeting PTPs are often hampered by off-target effects from nonselective binding of inhibitors. While Sts-1 is a protein tyrosine phosphatase, it is structurally distinct from canonical PTPs (20). To determine the selectivity of the inhibitors for Sts-1 HP , we quantified the effect of a tetracycline derivative and an azo dye on the activity of the non-receptor type PTPs PTP1B and SHP1.…”

“…two conserved histidine residues and formation of a phosphor-histidine intermediate (19,(21)(22)(23)(24). While the Sts proteins are known to catalyze the dephosphorylation of phosphotyrosines on protein substrates, they are structurally, functionally and mechanistically distinct from canonical protein tyrosine phosphatases (PTPs) such as PTP1B and SHP1 (5,20). An established substrate of both Sts-1 and Sts-2, in vivo, is the kinase Zap-70 (4,5,25).…”

“…Despite the potential as an immunostimulatory target, little has been reported regarding drug discovery or development. X-ray crystal structures, and complementary biochemical data, suggest that Sts-1 has a welldefined and seemingly druggable active site that is structurally unlike other PTPs or PGMs (19,20). To date, however, no specific inhibitors of either Sts-1 or Sts-2 have been reported.…”

“…To date, however, no specific inhibitors of either Sts-1 or Sts-2 have been reported. These proteins can be inhibited with the general competitive inhibitor, orthovanadate, and the SHP-2 inhibitor PHPS1 (4-[2-[1,5-dihydro-3-(4-nitrophenyl)-5-oxo-1-phenyl-4Hpyrazol-4-ylidene]hydrazinyl]-benzenesulfonic acid) inhibits Sts-1 with a K i of 1 μM (20,26,27).…”