The Trypanosoma brucei life cycle switch TbPTP1 is structurally conserved and dephosphorylates the nucleolar protein NOPP44/46

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.

FIGURE 1.

Substrate trapping identifies NOPP44/46 as a major TbPTP1 substrate. A, anti-Tyr(P) (α-pTyr) Western blot after TbPTP1 substrate trapping. The trapping mutant (trap) selectively enriches three major tyrosine-phosphorylated proteins. WT, wild type. B, anti-NOPP44/46 Western blot of proteins bound to wild type and trapping mutant. The Western blot in B is an experimental replicate of A and identifies the ∼45-kDa band as NOPP44/46. Some phospho-independent binding is also apparent. C, Coomassie Blue staining showing equivalent amounts of TbPTP1 wild type and mutant elute from the resin prior to trapping.

FIGURE 2.

TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. A TAP-tagged allele of NOPP44/46 was expressed in procyclic forms and affinity-purified for dephosphorylation reactions. A, TbPTP1 dephosphorylates NOPP44/46 in a dose-dependent manner. α-pTyr, anti-Tyr(P) antibody. B, TbPTP1, but not unrelated phosphatases from M. tuberculosis (PtpA and PtpB), S. aureus (SaPtpA), and L. monocytogenes (lmo1935), dephosphorylates NOPP44/46.

FIGURE 3.

NOPP44/46 is phosphorylated on Tyr¹⁸¹. A, schematic of NOPP44/46 indicating domain organization and position of the five tyrosine residues. U, unique region; J, junction; A, acidic region; R, RGG repeat region. B, all five NOPP44/46 tyrosines were individually changed to Phe, and the phosphorylation of NOPP44/46 was detected by anti-Tyr(P) antibody (α-pTyr) (upper panel) and anti-NOPP44/46 control Western (lower panel). WT, wild type.

FIGURE 4.

Overall structure of TbPTP1. A, TbPTP1 shares the canonical PTP fold. The catalytic motifs P-loop and WPD loop are highlighted in orange and yellow, respectively. No electron density for residues 65–74 was visible in chain A (dotted line). N-term, N terminus; C-term, C terminus. B, 2F_o − F_c electron density map of the active site showing phosphate (center), contoured at 1.0 σ.

FIGURE 5.

TbPTP1 has a similar fold to human PTPs. A, trypanosome-specific sequence motifs (green) map on the surface outside of the active site (P-loop in orange). B, superposition of the Cα chain in ribbon representation showing overall strong similarity to human PTPRO.

FIGURE 6.

Electrostatic surface potential of TbPTP1. A, left, the TbPTP1 surface shows distinct electronegative (red) and positive (blue) regions, with a continuous electropositive stretch across the active site. The entry to the active site is indicated by the circle. Right, schematic representation in the same orientation as left panel. B, electrostatic surface representation of PTP1B (1SUG, left) and PTPRO (2G59, right) in the same orientation as TbPTP1. PTP1B also binds a highly negatively charged substrate and shows large electropositive areas.