Abstract
Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.
Highlights
T. brucei alternates between human and tsetse fly hosts, requiring extensive and rapid physiologic adaptations
Tyrosine phosphorylation was preserved throughout cell lysis by the addition of iodoacetamide and sodium orthovanadate to inhibit endogenous protein-tyrosine phosphatase (PTP)
The wildtype and D199A T. brucei protein-tyrosine phosphatase 1 (TbPTP1) variants were covalently linked to NHS-Sepharose beads and incubated with T. brucei lysate
Summary
Protein Expression, and Purification—The fulllength TbPTP1 (systematic ID Tb10.70.0070) gene was amplified from genomic T. brucei DNA (kindly provided by Dr Christian Klotz) and cloned into the pET28b expression vector in-frame with the N-terminal six-histidine tag. Fractions were pooled, loaded on a gel filtration column, and eluted in 20 mM Tris, pH 7.5, 100 mM NaCl. Recombinant TbPTP1 was concentrated to 10 mg/ml. Expression of the tagged protein was induced with tetracycline for 24 h, and the protein was purified using a modified tandem affinity purification protocol with 1 mM sodium orthovanadate in the lysis buffer [15, 16]. Cells were extracted in lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 1% Triton X-100) containing 1 mM sodium orthovanadate, complete protease inhibitor mixture (Roche Applied Science), and 5 mM iodoacetamide to inhibit endogenous PTP activity. The resin was boiled in reducing SDS-PAGE buffer for 15 min, run on a 12% Tris-glycine gel, and transferred to nitrocellulose membrane for Western analysis with 4G10 anti-Tyr(P) (GE Healthcare) and anti-NOPP44/46 antibody [9]. The crystal structure was deposited in the Protein Data Bank under accession number 3M4U
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