Browsing by Author "Song, Wei"

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    Structural basis for sulfatide recognition by Disabled-2
    Song, Wei (Virginia Tech, 2021-01-12)
    Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy.
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    Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides
    Song, Wei; Gottschalk, Carter J.; Tang, Tuo-Xian; Biscardi, Andrew; Ellena, Jeffrey F.; Finkielstein, Carla V.; Brown, Anne M.; Capelluto, Daniel G. S. (2020-08-11)
    Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin alpha (IIb)beta (3) receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin alpha (IIb)beta (3) receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.