The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain.
The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain.
复制标题
钙网蛋白的钙和锌响应区域严格位于 N-/C-结构域。
DOI：
10.1016/j.bbagen.2006.02.003
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Bouvier,Marlene
中科院分区：
文献类型：
--
作者：
Tan,Yan;Chen,Mingnan;Li,Zhenjie;Mabuchi,Katsuhide;Bouvier,Marlene
钙网蛋白（CRT）是内质网的伴侣。我们将CRT分为两个结构域，N-/C-结构域和P-结构域，以确定其金属离子响应区域。为此，我们构建了N-/C-结构域（1-180通过接头融合到290-400）和P-结构域（189-280）的细菌表达系统。圆二色性（CD）研究表明，钙离子增加三级包装和热稳定性的载脂蛋白N-/C-结构域，而锌离子有很强的去稳定作用。有趣的是，钙离子和锌离子都没有改变载脂蛋白P结构域的结构特性。这些结果表明，钙和锌的响应区域严格驻留在N-/C-域。CRT、N-/C-结构域和P-结构域的热变性曲线的分析表明P-结构域在CRT中的结构作用。CRT和钙连接蛋白的旋转阴影电子显微镜（EM）分析为它们的结构相关性提供了令人信服的证据。该分析还显示，载脂蛋白P-结构域采用各种弯曲形状，表明构象灵活性。载脂蛋白N-/C-结构域的EM图像显示具有宽间隙的对象，表明N-和C-结构域之间的弱相互作用。这与凝胶过滤柱上apo N-/C-结构域的较大尺寸一致。我们的研究提供了一个框架相关的CRT的结构组织与其金属离子响应区域。
Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1–180 fused by a linker to 290–400) and P-domain (189–280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region.