Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila

Abstract

Legionnaires' disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an α-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer whose dimer interface involves interactions between the coiled coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric state to a heterodimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors.

Keywords: Legionella effector; Michaelis-Menten; coiled-coil; crystal structure; enzyme kinetics; phosphatase; phosphoesterase fold; tyrosine phosphatase; tyrosine-protein phosphatase (tyrosine phosphatase).

Figure 1.

Purified WipA proteins. Left panel, SDS-PAGE of purified WipA fragments. Right panel, table with the corresponding molecular masses.

Figure 2.

The WipA structure and folding topology. A, cartoon representation of the WipA435 structure in two orientations rotated by 180°. Color coding is based on the secondary structure elements and domains: α-helical hairpin in red, β-sheet linking the hairpin to the phosphatase domain in brown, remaining β-strands in green, and α-helices in yellow. The N and C termini of the protein and the secondary structure elements are labeled. The manganese ion is represented as a purple sphere, and the two coordinated water molecules are small red spheres. Key residues highlighting the active center are represented as sticks. In the panel at right, the edge strands β7 and β8 are clearly visible: they contain residues 411–422, i.e. the region missing in WipA411 but present in WipA435. B, the WipA435 folding topology. Color codes are as in A. The two central β-sheets are highlighted by dashed boxes. The positions of the phosphoesterase motifs residues are labeled as solid black circles. The Arg³⁶⁹ involved in the phosphotyrosine specificity is indicated with a white circle.

Figure 3.

Structural comparison of WipA with other phosphatases. A, cartoon representation of superposed structures from WipA in same color coding as in Fig. 2 and the CAPTPase from S. spongiae in cyan (PDB code 1v73). Secondary structure elements of WipA common to both WipA and CAPTPase are labeled according to Fig. 2 (the conserved α5-helix is not shown in this orientation). B, cartoon representation of superposed WipA (color-coded and oriented as in A) and the human serine-threonine phosphatase PP1α in magenta (PDB code 3e7b). Secondary structure elements of WipA common to both WipA and PP1α are labeled (the conserved α5-helix is not shown in this orientation). C, comparison of an archetypical protein-tyrosine phosphatase, PTP1β (PDB code 1pty) (left panel) and WipA (right panel). Color coding of WipA is as in Fig. 2, whereas for PTP1β, color coding is yellow for helices and green for strands.

Figure 4.

WipA domain interfaces. A, cartoon representation of the interface between the phosphatase domain and the α-helical hairpin in WipA. Key secondary structure elements of WipA are labeled, and color coding is as in Fig. 2. The interaction is based on an H-bond and salt-bridge network that is highlighted by dashed lines. B, cartoon representation of the functionally relevant dimer of WipA. This dimer is found in both WipA411 and WipA435 crystals. It is mediated by interaction between the coiled-coil region of the α-helical hairpin and the globular phosphatase domain, which was shown to be the relevant dimer in solution (see main text and C). C, SEC-MALS analysis of various WipA fragments. The inset reports on the MW measured experimentally using SEC-MALS and the molecular mass calculated from the sequence.

Figure 5.

WipA active site and phosphopeptide specificity. A, cartoon representation of the active site following the color-coding of Fig. 2. The amino acids of the phosphoesterase motif are labeled, and H-bond interactions are represented by dashed lines. Distances for those bonds are reported. B, dephosphorylation of phosphotyrosine and phosphothreonine-containing peptides by WipA. Time courses of hydrolysis are shown using 20 nm full-length wild-type WipA. C, dephosphorylation of the phosphotyrosine peptide END(pY)INAS by WipA and WipA mutants. Phosphate release is plotted against increasing concentrations of WipA proteins. D, dephosphorylation of the phosphothreonine peptide RRA(pT)VA by WipA and WipA mutants. Phosphate release is plotted against increasing concentrations of WipA proteins. E, dephosphorylation of tyrosine-phosphorylated FGFR3 by WipA. The extent of tyrosine-phosphorylation of residue Tyr⁷⁶⁰ and Tyr⁶⁴⁸ in the FGFR3 kinase domain was monitored by Western blotting analysis followed by specific detection of the phosphorylation state of these residues using specific anti-Tyr(P)⁷⁶⁰ (α-pY760) and anti-Tyr(P)⁶⁴⁸ (α-pY648) antibodies (indicated for each panel). Left panel, from left to right the blots indicate the phosphorylation levels of the FGFR3 Tyr⁷⁶⁰ incubated at WipA concentrations of 0, 40, 100, and 140 nm; a control 140 nm WipA Arg³⁶⁹ is shown. Middle panel, same as in left panel but for phosphorylation levels of the FGFR3 Tyr⁶⁴⁸. Right panel, time course of phosphorylation levels of the FGRF3 Tyr⁷⁶⁰ incubated with 60 nm WipA at 0, 1, 2, 5, and 10 min.