PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders

Abstract

Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.

Keywords: Auto-inflammation; Immunology; LYP; PSTPIP1.

Fig. 1

Interaction of LYP with of PSTPIP1 mutants associated to immune diseases. A Domain structure of PSTPIP1 with the mutations studied on this work. Mutations described in patients suffering from autoinflammatory diseases and CVID are indicated on top, while W232A mutation that is not related to disease is on the lower part. B HEK293 cells were transfected with PSTPIP1-V5 wild type and several mutants associated with autoinflammatory diseases, as indicated in the top of the panels, and with 3xmyc-LYP. Lysates were immunoprecipitated with myc antibody (Ab) and PSTPIP1 bound to LYP was detected by IB with Ab for the V5 epitope. Expression of the proteins was verified by IB in total lysates (TL) with the same Abs. C Dissociation constants (k_d) of the interaction of the F-BAR domain of PSTPIP1, wild type and indicated mutants, with fluorescein-LYP-CTH. Data are means ± standard deviation (SD), n = 3 independent experiments, one shown in D. Statistical comparison to the k_d of the wild type F-BAR was analyzed using ANOVA followed by Dunnett’s test. ****p < 0.0001. D Representative binding isotherms of the F-BAR domain of PSTPIP1, wild type and mutants, to fluorescein-labeled LYP-CTH (2 nM), measured by fluorescence anisotropy. Data points are means ± SD of measurement replicates; lines represent the fitted binding curves. E HEK293 cells co-transfected with LYP-mCherry (red) and wild type or indicated mutants of PSTPIP1-eGFP (green). Manders’ colocalization indexes (M2) were calculated. Error bars represent SEM (n > 10). **p < 0.01; ****p < 0.0001, by Student’s t test

Fig. 2

Structure of the F-BAR domain of PSTPIP1. (A) Two orthogonal views of a ribbon representation of the structure of PSTPIP1. Protomers of the dimer are colored in orange and blue, respectively. (B) Surface representation of the F-BAR dimer colored by the electrostatic potential. Clusters of basic residues in the concave surface are encircled with dashed lines. Acidic and basic residues on the surface are labeled in one of the protomers

Fig. 3

Structural determinants of the PSTPIP1/LYP interaction. A Orthogonal views of the overall structure of the complex. The two protomers of the PSTPIP1 dimer, A (orange) and B (blue), and LYP (magenta) are colored similarly throughout the figure. B Interaction of LYP, shown as sticks, with PSTPIP1, shown as surface. Residues of PSTPIP1 that participate in the interface are labeled. The three hydrophobic pockets of the binding site are highlighted by dashed-line circles. C Two views showing contacts between LYP and PSTPIP1. H-bonds and salt bridges are shown as dashed lines. D Sequence alignment of the regions of human PSTPIP1 and PSTPIP2 (Uniprot Q9H939) that contain the LYP-binding site. Residues from protomers A and B, which form the interface, are indicated by inverted orange triangles and blue circles, respectively. Residues whose solvent exposed surface was buried ≤ 20% by LYP are indicated by open symbols. Conserved residues are shown in green boxes. E Multiple sequence alignment of the CTH regions of phosphatases LYP, PTP-PEST (Uniprot Q05209) and PTPN18 (Uniprot Q99952). Residues of LYP that make specific contacts with PSTPIP1 are indicated by inverted triangles. Conserved residues are shown in green boxes. (F) His-PSTPIP1 full- length recombinant protein was subjected to pulldown assays with the indicated CTH peptide fused to GST. The presence of PSTPIP1 in the precipitates was visualized by IB with antibody against PSTPIP1, and GST–peptides were detected with an antibody against GST

Fig. 4

Interaction of LYP with F-BAR dimers. A, B HEK293 cells were transfected with PSTPIP1 F-BAR dimers in 3xmyc plasmid, as indicated in the top of the panels, and with 3xFLAG-LYP. Lysates were immunoprecipitated with myc Ab and LYP bound to PSTPIP1 was detected by IB with Ab for LYP. Expression of the proteins was verified by IB in total lysates (TL) with the same Abs. C Determination of the interaction stoichiometry by titration of fluorescein-LYP-CTH (1 µM) with the F-BAR of PSTPIP1 (residues 1–289) using fluorescence anisotropy. The stoichiometry (N) was determined from the intersection of asymptotic lines in the linear ascending and saturation (i.e. plateau) regions. D ITC analysis of LYP binding to PSTPIP1. Upper panel, heat exchange of the dilution of the unlabeled LYP-CTH peptide in buffer 20 mM sodium phosphate (pH 7.5), 150 mM NaCl, 5% (v/v) DMSO (upper trace) and representative thermogram of the binding of LYP-CTH to the F-BAR of PSTPIP1 (lower trace). Lower panel, LYP binding isotherm to PSTPIP1. The red line is the fit of a one binding site model to the heat changes of the interaction after subtracting the heat change of the dilution of LYP in buffer. The reported values of N, k_d, and binding enthalpy (ΔH) are the means ± standard deviations of three independent experiments

Fig. 5

Pathogenic mutations of PSTPIP1. A Surface representation of the structure of PSTPIP1. Residues mutated in autoimmune diseases are shown in red and blue for each protomer of the F-BAR dimer, respectively. The position of W232 is also indicated. B HEK293 cells were transfected with several PSTPIP1 mutants fused to EGFP, as indicated in the top of the panels, and with PSTPIP1-myc. Lysates were immunoprecipitated with anti-myc Ab and the different mutants of PSTPIP1-EGFP precipitated with PSTPIP1-myc were detected by IB with anti-GFP Ab. Expression of the proteins was verified by IB in total lysates (TL) with the same Abs