Conformational Analysis of Selectins

描述(申请人提供)：选择素是重要的分子，通过与相对细胞上的碳水化合物配体结合，调节白细胞和内皮细胞之间的细胞-细胞接触。这些接触会导致白细胞滚动，这是炎症反应的早期步骤。抑制这些相互作用为治疗包括关节炎、牛皮癣和转移在内的多种疾病提供了一种可能的方法。选择素在滚动过程中经历了很大的构象变化，从而改变了它们对碳水化合物的亲和力。这里提出了一个高亲和力构象的P-选择素(由Springer实验室的Uyen Phan创建)的晶体结构将在有和没有结合配体的情况下被解决。同时，L-选择素的结晶将是一个近期的目标，因为L-选择素没有结构，而且不同的选择素之间存在底物特异性差异。锁定在低亲和力构象中的P-选择素将通过二硫键的加成来构建，并在有或没有结合碳水化合物的情况下结晶。流室分析将被用来评估这种低亲和力构象与各种底物的结合。这项工作将深入了解滚动的机制，以及碳水化合物结合界面的重要结构特征，这可能是抑制剂的目标。

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.