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E-钙黏蛋白结构域的钙结合与同源缔合

Calcium binding and homoassociation of E-cadherin domains.

作者信息

Koch A W, Pokutta S, Lustig A, Engel J

机构信息

Department of Biophysical Chemistry, Biozentrum, University of Basel, Switzerland.

出版信息

Biochemistry. 1997 Jun 24;36(25):7697-705. doi: 10.1021/bi9705624.

Abstract

Cadherins are single pass transmembrane glycoproteins which mediate calcium dependent cell-cell adhesion by homophilic interactions. To reveal the molecular details of calcium binding and homoassociation, we recombinantly expressed in Escherichia coli a domain pair consisting of the first two domains of E-cadherin (ECAD12) and the single domains 1, 2, and 5. ECAD12 encompasses the most N-terminal of the four putative calcium-binding pockets in the extracellular region of E-cadherin. Equilibrium dialysis experiments revealed that the single domains do not bind Ca2+, but ECAD12 was found to bind three calcium ions. ECAD12 dimerizes (Kd = 0.08 +/- 0.02 mM) in the presence of Ca2+ as we could demonstrate by analytical ultracentrifugation. Calcium binding to ECAD12 induces conformational changes which were monitored by electrophoretic mobility and by circular dichroism. By analyzing our equilibrium dialysis data with a single binding site model, we found an average Kd of 460 microM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical Kds of 330 microM and a third much higher Kd of 2 mM. The entire extracellular region of E-cadherin, which was recombinantly expressed in mammalian cells, binds nine Ca2+ with a much lower average Kd of 30 microM. Therefore, we conclude that the four calcium binding pockets are not identical. Since binding to ECAD12 occurs at Ca2+ concentrations close to those in the extracellular space, we suggest that the N-terminal domain pair might be involved in calcium regulation of E-cadherin mediated cell-cell adhesion.

摘要

钙黏蛋白是单次跨膜糖蛋白,通过同源相互作用介导钙依赖性细胞间黏附。为了揭示钙结合和同源缔合的分子细节,我们在大肠杆菌中重组表达了由E-钙黏蛋白的前两个结构域(ECAD12)以及单个结构域1、2和5组成的结构域对。ECAD12包含E-钙黏蛋白细胞外区域四个假定钙结合口袋中最N端的口袋。平衡透析实验表明,单个结构域不结合Ca2+,但发现ECAD12结合三个钙离子。正如我们通过分析超速离心所证明的,在Ca2+存在的情况下,ECAD12二聚化(Kd = 0.08 +/- 0.02 mM)。钙与ECAD12的结合会诱导构象变化,通过电泳迁移率和圆二色性进行监测。通过用单结合位点模型分析我们的平衡透析数据,我们发现三个结合的Ca2+的平均Kd为460 microM。假设一个三个结合位点的模型,这稍微提高了拟合质量,我们得到两个相同的Kd为330 microM,第三个Kd高得多,为2 mM。在哺乳动物细胞中重组表达的E-钙黏蛋白的整个细胞外区域结合九个Ca2+,平均Kd低得多,为30 microM。因此,我们得出结论,四个钙结合口袋并不相同。由于与ECAD12的结合发生在接近细胞外空间的Ca2+浓度下,我们认为N端结构域对可能参与E-钙黏蛋白介导的细胞间黏附的钙调节。

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