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 LewisX and sialyl LewisA (sLeX/A). Existing crystal structures of the extracellular lectin/EGF-like domain of E-selectin complexed with sLeX 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.

Insights into Selectin Inhibitor Design from Endogenous Isomeric Ligands of SLea and SLex

“…In the E-selectin–sLe x crystal structure, the NAc group at the C2 position of GlcNAc is in close proximity to a loop region (residues 80 to 89) that has been demonstrated to be critical for the binding of sLe x to E-selectin (Figure ). This loop region displayed a closed conformation found in the cocrystal complex that locks the oligosaccharide in the binding site, but an open conformation in a soaked complex, where the binding interactions were considered to be weaker. ,, Theoretical studies also suggested that the conformation for this loop could change from open to close after sLe x lodging into the binding site …”

“…This loop region displayed a closed conformation found in the cocrystal complex that locks the oligosaccharide in the binding site, but an open conformation in a soaked complex, where the binding interactions were considered to be weaker. 25,42,65 Theoretical studies also suggested that the conformation for this loop could change from open to close after sLe x lodging into the binding site. 66 The representative structure of the E-selectin−sLe x complex from MD simulations showed that the NAc group resided between the side chains of R84 and Q85 (Figure 2A,B), while a smaller ethylene glycol group was in the same location present in the representative structure for the E-selectin−sLe a complex (Figure 2A,C).…”

“…11−20 Selectins also play an important role in cancer progression as they function in the cancer cell adhesion to endothelia in metastasis. 10,21−23 All three selectins bind to glycoproteins and glycolipids with a common α2−3-sialylated and α1,3/4-fucosylated carbohydrate epitope, sialyl Lewis x (sLe x , Neu5Acα2−3Galβ1−4[Fucα1− 3]GlcNAcβ1-R) (Figure 1) and sialyl Lewis a (sLe a , Neu5Acα2−3Galβ1−3[Fucα1−4]GlcNAcβ1-R), carried by mucin-type N-and O-linked oligosaccharides 24,25 and found on most leukocytes and some endothelial cells. 21,26−28 In addition to their innate tethering function as the counterpart for selectins, both sLe a and sLe x are also epitopes of antigens that have been widely used as tumor markers in the clinical diagnosis of pancreatic, gastrointestinal, and breast cancers.…”

“…Selectins are a family of three Ca 2+ -dependent C-type lectins present on the surface of numerous cell types in the vasculature, including endothelial cells (E-selectin), platelets (P-selectin), and leukocytes (L-selectin). − They interact with the cell-surface glycans to promote the initial tethering and rolling of leukocytes on the surface of endothelial cells and, thus, play an essential role in the recruitment of leukocytes and the inflammation process. − Inhibiting their interactions with the endogenous glycan counterparts is promising for neutralizing excessive inflammatory responses in many cardiovascular and immune dysfunctions. − Selectins also play an important role in cancer progression as they function in the cancer cell adhesion to endothelia in metastasis. ,− All three selectins bind to glycoproteins and glycolipids with a common α2–3-sialylated and α1,3/4-fucosylated carbohydrate epitope, sialyl Lewis x (sLe x , Neu5Acα2–3Galβ1–4­[Fucα1–3]­GlcNAcβ1-R) (Figure ) and sialyl Lewis a (sLe a , Neu5Acα2–3Galβ1–3­[Fucα1–4]­GlcNAcβ1-R), carried by mucin-type N - and O -linked oligosaccharides , and found on most leukocytes and some endothelial cells. ,− In addition to their innate tethering function as the counterpart for selectins, both sLe a and sLe x are also epitopes of antigens that have been widely used as tumor markers in the clinical diagnosis of pancreatic, gastrointestinal, and breast cancers. − Both sLe a and sLe x are constitutional isomers as they have exactly the same chemical compositions, but differ in the glycosidic linkages, where Fuc and Gal exchange their linkage positions to GlcNAc in sLe x and sLe a . Counterintuitively, these distinct linkage differences do not alter the relative positions of Fuc and Gal, as well as Neu5Ac, and only result in orientational changes for GlcNAc residue.…”