Protein Signaling Domains Laboratory, Department of Biological Sciences, Fralin Life Sciences Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg, VA, 24061, USA.
Research and Informatics, University Libraries, Biochemistry Department, and Center for Drug Discovery, Virginia Tech, Blacksburg, 24061, VA, USA.
Sci Rep. 2020 Aug 11;10(1):13520. doi: 10.1038/s41598-020-70478-0.
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 αβ 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 αβ 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.
Disabled-2 (Dab2) 是一种衔接蛋白，通过两种机制调节血小板聚集的程度。在第一种机制中，Dab2 在细胞内下调整合素 αβ 受体，将其转化为低亲和力状态，以利于黏附和聚集过程。在第二种机制中，Dab2 被释放到细胞外，并与促聚集介质（整合素 αβ 受体和硫酸脑苷脂）相互作用，分别阻断它们与纤维蛋白原和 P 选择素的结合。我们之前的研究表明，Dab2 内的一个 35 个氨基酸区域，我们称之为硫酸脑苷脂结合肽（SBP），包含两个潜在的硫酸脑苷脂结合基序，由两个连续的多碱性区域表示。本研究通过分子对接、核磁共振、脂质结合测定和表面等离子体共振，确定了 SBP 中负责硫酸脑苷脂结合的关键 Dab2 残基。分子对接表明，一个主要由 R42 介导的亲水区域负责与硫酸脑苷脂的头部基团相互作用，而 C 末端多碱性区域有助于与酰基链相互作用。此外，我们证明了在 Dab2 SBP 中，R42 显著抑制血小板 P 选择素表面表达。与硫酸脑苷脂相互作用的 Dab2 SBP 残基与其他蛋白质中描述的神经鞘脂结合残基相似，表明硫酸脑苷脂结合蛋白具有共同的结合机制。
