Stabilization of the Hinge Region of Human E-selectin Enhances Binding Affinity to Ligands Under Force

Abstract

Introduction: E-selectin is a member of the selectin family of cell adhesion molecules expressed on the plasma membrane of inflamed endothelium and facilitates initial leukocyte tethering and subsequent cell rolling during the early stages of the inflammatory response via binding to glycoproteins expressing sialyl Lewis^X and sialyl Lewis^A (sLe^X/A). Existing crystal structures of the extracellular lectin/EGF-like domain of E-selectin complexed with sLe^X have revealed that E-selectin can exist in two conformation states, a low affinity (bent) conformation, and a high affinity (extended) conformation. The differentiating characteristic of the two conformations is the interdomain angle between the lectin and the EGF-like domain.

Methods: Using molecular dynamics (MD) simulations we observed that in the absence of tensile force E-selectin undergoes spontaneous switching between the two conformational states at equilibrium. A single amino acid substitution at residue 2 (serine to tyrosine) on the lectin domain favors the extended conformation.

Results: Steered molecular dynamics (SMD) simulations of E-selectin and PSGL-1 in conjunction with experimental cell adhesion assays show a longer binding lifetime of E-selectin (S2Y) to PSGL-1 compared to wildtype protein.

Conclusions: The findings in this study advance our understanding into how the structural makeup of E-selectin allosterically influences its adhesive dynamics.

Keywords: Conformational states; E-selectin; Molecular dynamics; Selectin ligand binding dynamics; Steered molecular dynamics.

Figure 1

E-selectin spontaneously transitions from bent to extended conformational state and back again. (a) MD trajectory representations of E-selectin during the least displacement and the highest displacement, respectively. (b) PC1 vs PC2 shows the predicted transitioning path of E-selectin from bent, to extended, and back to bent. (c) RMSDs of MD simulations as a function of time. (d) Angle measurement of the interdomain hinge region as a function of time. (e) Ribbon representations of E-selectin and the measured angle at t = 10 ns and t = 30 ns. (Blue lectin domain, Pink EGF -like domain).

Figure 2

Dynamic hydrogen network stabilizes the interdomain hinge region. Representative illustrations of human E-selectin MD simulations at indicated time points showing the formation and breakage of hydrogen bond network as the protein transitions from the bent to the extended conformation and back again at the interdomain region. Yellow dashed lines represent hydrogen bonds; Red arrows indicate trajectories of indicated residues.

Figure 3

Point mutation of residue 2 from Serine to Tyrosine eliminates the spontaneous transition between the two conformational states.

Figure 4

High affinity extended conformation allosterically forces the binding pocket to also adopt a high-affinity conformation in E-selectin. (a) RMSF per residue of the lectin domain of wildtype E-selectin (W/T) and variant E-selectin (S2Y). Grey rectangle highlights the flexible loop containing residues 83-85 of the binding pocket. (b) Schematics of flexible loop outlining the reduction of fluctuation in E-selectin (WT) compared to E-selectin (S2Y).

Figure 5

SMD simulations show PSGL-1 has higher affinity to E-Selectin (S2Y) than E-Selectin (W/T). (a) SMD simulation showing the dissociation time (in simulation time) of PSGL-1 from E-Selectin. (b) Representative illustrations of human E-selectin interaction with PSGL-1 as it is being pulled away from E-Selectin at the indicated time points. E-selectin is blue; PSGL-1 is grey. Purple lines are representative of inter-molecular interactions.

Figure 6

E-selectin (S2Y) has increased adhesiveness to ligands compared to the wildtype. The average rolling velocity of model cell line, KG-1a, ligands rolling on microtubes coated E-selectin-Fc chimeric protein (filled black square wildtype, filled red square S2Y, filled green square S2W, filled blue square S2C; n = 3).