Structure, sulfatide binding properties, and inhibition of platelet aggregation by a disabled-2 protein-derived peptide

Abstract

Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α(IIb)β(3) integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.

FIGURE 1.

Dab2 SBM is disordered in aqueous solution but becomes structured in the presence of DPC micelles. A, sequences of the closest homologues of Dab proteins corresponding to the SBM region. The two sulfatide-binding sites identified in human Dab2 (hDab2) are bolded, and the key lysine residues are boxed. mDab2, mouse Dab2. B, far-UV CD spectra of Dab2 SBM in the absence (dotted line) and presence (solid line) of DPC micelles. deg, degree. C, two-dimensional ¹H, ¹⁵N HSQC of uniformly ¹³C, ¹⁵N-labeled Dab2 SBM in DPC micelles. Resonances of the backbone amides are labeled according to human Dab2 protein sequence. Asterisks on Gly-31, Glu-33, and Lys-34 resonances likely are due to cis/trans isomerization of Pro-32. MRE, mean residue ellipticity.

FIGURE 2.

NOE patterns and NMR structure of micelle-bound Dab2 SBM. A, summary of the sequential and medium range NOE connectivities for micelle-bound Dab2 SBM. The thickness of the sequential NOEs corresponds to the intensity of the NOE interaction between residues. B, the ensemble of the 20 energy-minimized conformers of micelle-bound Dab2 SBM. C, two views of a representative structure of Dab2 SBM with the two helical elements labeled in red. D, electrostatic surface representation of Dab2 SBM in two 180° orientations. Images in C and D were generated using PyMOL.

FIGURE 3.

Dab2 SBM binds sulfatides. A, overlay of ¹H, ¹⁵N HSQC of DPC-bound Dab2 SBM in the absence (black) and presence (red) of DPC-bound sulfatides. Perturbed chemical shifts are boxed and labeled with the corresponding residue. B, histogram representing the normalized chemical shift changes of Dab2 SBM induced by sulfatides. The colored bars represent significant perturbations: red (Δδ_average + 1 × S.D.) > yellow (Δδ_average). C, Dab2 SBM amino acids that display significant chemical shift changes are labeled and color-coded according to the scale defined in B. D, binding plot of the Dab2 SBM to sulfatide liposomes using SPR analysis. A. U., arbitrary units.

FIGURE 4.

Paramagnetic quenching of Dab2 SBM in the presence of sulfatide-embedded DPC micelles. A–C, intensity retention plots of the HSQC spectrum of Dab2 SBM in the presence of MnCl₂ (A), 5-DSA (B), and 16-DSA (C).

FIGURE 5.

Dab2 SBM precludes sulfatide-mediated platelet aggregation. Representative time series of images of test microchannels with platelets and aggregates under physiological flow conditions are indicated on top. Washed platelets loaded without (A) and with (B) sulfatide liposomes were preincubated with the following proteins/peptides (10 μm each): Dab2 N-PTB (C), Dab2 N-PTB^4M (D), Dab2 SBM (E), and RGDS (F). Images were taken 0, 3, and 10 min after the start of flow. The last column on the right illustrates how unadhered objects (green), isolated platelets (white), and platelet aggregates (red) are visualized in the channel. Scale bar, 45 μm; arrows indicate flow direction. Representative of n = 3 experiments. Lipo-S, sulfatide-enriched liposomes.

FIGURE 6.

Modulation of the number of platelet aggregates by Dab2 SBM. A, bar graphs representing the average count of platelet aggregates found in four different fields on the microchannel at 500 s. Washed platelets were flowed over a microchannel in the absence of ADP and sulfatides or stimulated with ADP and preincubated with sulfatide liposomes. In addition, ADP-stimulated sulfatide-treated platelets were preincubated with the following proteins/peptides (10 μm each): Dab2 N-PTB, Dab2 N-PTB^4M, RGDS, and Dab2 SBM. Samples were equilibrated for at least 2 min at 70 s⁻¹ before data collection. B, bar graphs depicting the average area of platelet clusters of four different regions of the microchannel with the experimental conditions described in A. In both panels, * indicates p < 0.05 when compared with the ADP + Lipo-S condition. Error bars indicate S.D. Lipo-S, sulfatide-enriched liposomes.