Structural,in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides

Abstract

25Disabled-2 (Dab2) is an adaptor protein that regulates numerous cellular processes. Among them, Dab2 26 modulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 27 intracellularly downregulates the integrin aIIbb3 receptor, converting it to a low affinity state for adhesion 28 and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with 29 both the integrin aIIbb3 receptor and sulfatides, both of which are known to be pro-aggregatory mediators, 30 blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 31 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two 32 potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using a combined 33 methodology including molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface 34 plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for 35 sulfatide binding. A hydrophilic region, primarily mediated by R42, is responsible for the interaction with 36 the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with the acyl 37 chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of 38 platelet P-selectin surface expression. The interacting Dab2 SBP residues with sulfatide resemble those 39 described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share 40 common binding mechanisms. 41 42 43 44 integrin receptor.45 46 3 INTRODUCTION 47 48The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of 49 cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and 50 modulation of platelet aggregation (1). Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 51 is primarily expressed in the brain (2), whereas Dab2 is ubiquitously expressed in different tissues (3, 4). 52Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and 53 progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate (5), bladder (6), and colorectal 54 cancer cells (7), suggesting that Dab2 can act as a tumor suppressor (8). Two alternative spliced forms of 55 Dab2 are expressed in humans, p96 and p67, with the latter lacking a central exon (3). Dab2 contains a 56 phosphotyrosine-binding (PTB) domain located at the N-terminus, clathrin-binding sites, NPF and DPF 57 motifs, and a proline-rich SH3 domain located at the C-terminus, indicating that Dab2 primarily functions 58 as an adaptor protein. 60Recently, Dab2 was characterized as a negative regulator of platelet aggregation by modulating both pro-61 aggregatory inside-out and outside-in signaling pathways (1). Modulation of the inside-out signaling by 62 Dab2 is mediated by its cytosolic S24-phosphorylated form, which b...

Utilization of computational techniques and tools to introduce or reinforce knowledge of biochemistry and protein structure–function relationships

“…The open pedagogy design approach to UR in biochemistry and utilization of computational techniques reinforces wet‐lab experiences and have been successful in creating meaningful poster presentations and publications. In using this approach, to date, we have supported UR students to present in over 40 poster sessions at 10 different local, national, and international conferences and be authors on 5 peer‐reviewed research articles in notable journals 5–9 . This short communication highlights an open pedagogy design approach to UR in biochemistry and the utilization of computational techniques and tools to both reinforce wet‐lab experiences and provide routes for experiential learning and workforce development when in‐person laboratory work is not feasible.…”