NMR structures of 36 and 73-residue fragments of the calreticulin P-domain
NMR structures of 36 and 73-residue fragments of the calreticulin P-domain
复制标题
DOI：
10.1016/s0022-2836(02)00812-4
复制
发表时间：
2002-09-27
影响因子：
5.6
通讯作者：
Wüthrich, K
中科院分区：
文献类型：
--
作者：
Ellgaard, L;Bettendorff, P;Wüthrich, K
钙网蛋白 (CRT) 是一种丰富的、可溶性的内质网分子伴侣。与其膜结合同源钙连接蛋白 (CNX) 类似，它是一种凝集素，可促进携带 N 连接聚糖的蛋白质折叠。这两种蛋白都与相关的共伴侣硫醇二硫化物氧化还原酶 ERp57 合作。该酶催化 CNX 和 CRT 结合糖蛋白底物中二硫键的形成。此前，我们解析了包含残基 189 - 288 的 CRT 中心富含脯氨酸的 P 结构域的 NMR 结构。该结构显示出扩展的发夹拓扑结构，具有三个短的反平行 β 折叠、三个小的疏水簇和发夹尖端的一个螺旋转角。我们进一步证明，CRT P 结构域顶端的残基 225-251 参与与 ERp57 的直接接触。在这里，我们表明CRT P结构域片段CRT（221-256）构成了一个自主折叠单元，并且具有与CRT（189-288）中相应区域高度相似的结构。 CRT(221-256) 中存在 36 个残基，其中 32 个形成结构良好的核心，使该片段成为已知最小的天然序列之一，可以在没有二硫键或紧密结合的金属离子的情况下形成稳定的非螺旋折叠。 CRT(221256) 包含完整 P 结构域的所有残基，显示与 ERp57 相互作用。等温滴定微量热法 (ITC) 现在显示该片段对 ERp57 的亲和力与完整 P 结构域的亲和力相似，表明 CRT(221-256) 可用作 CRT 的低分子量模拟物，用于进一步研究与 ERp57 的相互作用。我们还解析了 73 个残基片段 CRT(189-261) 的 NMR 结构，其中发夹尖端和第一个 β 折叠结构良好，但残基 189-213 是无序的，可能是由于发夹上缺乏稳定的相互作用。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189 - 288. This structure shows an extended hairpin topology, with three short anti-parallel beta-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225-251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221-256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189-288). Of the 36 residues present in CRT(221-256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221-256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189-261), in which the tip of the hairpin and the first beta-sheet are well structured, but the residues 189-213 are disordered, presumably due to lack of stabilizing interactions across the hairpin. (C) 2002 Elsevier Science Ltd. All rights reserved.