Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. Their extracellular structure is composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. Furthermore, selectins play a role beyond fixing cells to a specific location by regulating important signaling pathways in the migrating cell during physiological and pathological processes. These interactions start mainly with the binding of the lectin domain of selectins and ligand on cells. Therefore, structural/functional studies to date have mainly focused on the direct interactions of the lectin domain of E-selectin with its ligands while other domains and conformational dynamics received less attention. For this purpose, we produced a number of different recombinant E-selectin proteins with and without artificial oligomerization and with changes in the SCR units in addition to proteins where strategic residues will be mutated to change the conformation of the selectin to an extended conformer. Moreover, double cysteine mutant candidates were produced for maleimide labeling for the real-time SM-FRET (single molecule fluorescence resonance energy transfer) studies to assess conformational dynamics of E-selectin. Using a comprehensive set of static- and flow-based assays, we concluded that SCR domains play a role by enhancing the interaction of recombinant E-selectin proteins with E-selectin ligand, while dimerization and extension of the lectin domain improve the binding. However, our double cysteine mutants purification and labeling requires further optimization to be utilized to study the conformational dynamics of E-selectin binding to its ligands using SM-FRET and force microscopy. Furthermore, our experiments extend to highlight the importance of phosphatases in regulating signaling pathways that are affected by E-selectin binding to migrating cells. Collectively, these studies are beneficial to understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells.

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