Hong Zebin, Nowakowski Michal, Spronk Chris, Petersen Steen V, Andreasen Peter A, Koźmiński Wiktor, Mulder Frans A A, Jensen Jan K
*Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, 8000 Aarhus C, Denmark.
†Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 00-927 Warsaw, Poland.
Biochem J. 2015 Mar 1;466(2):299-309. doi: 10.1042/BJ20141236.
A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.
十年前，含八个半胱氨酸的N端基序（MANEC）被定义为一个新的蛋白质结构域家族。该结构域仅存在于来自动物的400多种多结构域I型跨膜蛋白的N端。尽管含有MANEC的蛋白质数量众多，但在蛋白质水平上仅对一种进行了表征：肝细胞生长因子激活剂抑制剂-1（HAI-1）。HAI-1是一种必需蛋白，因为基因敲除小鼠会因胎盘缺陷在子宫内死亡。HAI-1是matriptase、hepsin和肝细胞生长因子（HGF）激活剂的抑制剂，这些都是在上皮发育、细胞生长和体内平衡中起重要作用的丝氨酸蛋白酶。这些蛋白酶的失调与诸如皮肤病和癌症等病理状况有因果关系。由于缺乏关于MANEC的结构信息，对HAI-1和其他含MANEC的蛋白质的详细功能理解受到阻碍。尽管存在许多MANEC序列，但基于序列的数据库搜索无法预测结构同源性。在本文中，我们展示了HAI-1的MANEC结构域的核磁共振溶液结构，这是MANEC结构域家族的首个三维（3D）结构。出乎意料的是，MANEC是PAN/苹果结构域家族的一个新亚类，具有其自身统一的特征，例如两个额外的二硫键、两个延伸环区域和额外的α-螺旋元件。正如对其他含PAN/苹果结构域的蛋白质所显示的那样，我们提出MANEC结构域在分子内和分子间相互作用中具有类似的活性作用。该结构为进一步阐明HAI-1的功能提供了一个工具，也为研究其他含MANEC的蛋白质提供了参考。
