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

Abstract

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 α iib β 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 α iib β 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. Abbreviations CD Circular dichroism Dab2 Disabled-2 DPC Dodecylphosphocholine GST Glutathione S-transferase N-PTB N-terminus containing the phosphotyrosine-binding domain SBP Sulfatide-binding peptide SPR Surface plasmon resonance The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and modulation of platelet aggregation 1. Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 is primarily expressed in the brain 2 , whereas Dab2 is ubiquitously expressed in different tissues 3,4. Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate 5 , bladder 6 , and colorectal cancer cells 7 , suggesting that Dab2 can act as a tumor suppressor 8 .

The repertoire of protein-sulfatide interactions reveal distinct modes of sulfatide recognition

“…Mutagenesis in Dab2 residues K25, K49, K51, and K53 reduces sulfatide association, affecting Dab2-mediated platelet aggregation ( Drahos et al, 2009 ). Later, structural studies identified the minimal region within Dab2 that binds sulfatide (residues 24–58 of human Dab2) with a dissociation constant ( K D ) of ∼30–50 μM ( Table 1 ), inhibiting platelet-platelet interactions ( Xiao et al, 2012 ; Song et al, 2020 ). This region, which we define here as the Dab2 sulfatide-binding motif (SBM), contains an N-terminal disordered region followed by two short α-helices, which are involved in membrane insertion ( Xiao et al, 2012 ).…”

“…One of the features of SBDs is that they display low amino acid sequence homology ( Fantini et al, 2002 ) ( Figure 2A ). Backbone dynamics studies of a peptide representing Dab2 SBM indicate that its N- and C-termini are flexible, whereas a rigid structure is found within the α-helical regions, which is not altered by sulfatide ( Song et al, 2020 ). Combined structural and molecular dynamics simulation (MDS) studies refined the sulfatide-binding site in Dab2 SBM.…”

“…Combined structural and molecular dynamics simulation (MDS) studies refined the sulfatide-binding site in Dab2 SBM. Residues located upstream and on the first α-helix are engaged in sulfatide head group contacts with Dab2 R42 playing a primary role, although other residues nearby, including E33, Y38, and K44 are required for sulfatide association ( Figure 2B ) ( Song et al, 2020 ). Replacement of R42 with lysine reduces association to sulfatide-enriched lipid bilayers and impairs inhibition of P-selectin surface location in platelets ( Song et al, 2020 ).…”

“…Residues located upstream and on the first α-helix are engaged in sulfatide head group contacts with Dab2 R42 playing a primary role, although other residues nearby, including E33, Y38, and K44 are required for sulfatide association ( Figure 2B ) ( Song et al, 2020 ). Replacement of R42 with lysine reduces association to sulfatide-enriched lipid bilayers and impairs inhibition of P-selectin surface location in platelets ( Song et al, 2020 ). Thus, the stereochemistry of R42, and not necessarily the positive charge in it, is critical for sulfatide binding.…”

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

“…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 peerreviewed research articles in notable journals. [5][6][7][8][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.…”