Conformational Analysis of Selectins

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DESCRIPTION (provided by applicant): Selectins are important molecules in mediating cell-cell contacts between leukocytes and endothelial cells, by binding to carbohydrate ligands on opposing cells. These contacts lead to leukocyte rolling, an early step in the inflammatory response. Inhibiting these interactions provides a possible approach to treating a number of disorders including arthritis, psoriasis, and metastasis. The selectins undergo large conformational changes during the process of rolling that alter their affinity for carbohydrates. Here it is proposed that a crystal structure of the high affinity conformation of P-selectin (created by Uyen Phan in the Springer lab) will be solved with and without bound ligand. In parallel, crystallization of L-selectin will be an immediate goal, as there are no structures for L-selectin, and there are substrate specificity differences among the selectins. P-selectin locked in a low affinity conformation will be constructed by the addition of disulfide bonds, and crystallized, with and without bound carbohydrate. Flow chamber assays will be used to assess the binding of this low affinity conformation to various substrates. This work will give insight into the mechanism of rolling, and the important structural features of the carbohydrate binding interface, the likely target for inhibitors.