Ellgaard L, Holtet T L, Nielsen P R, Etzerodt M, Gliemann J, Thøgersen H C
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Eur J Biochem. 1997 Mar 1;244(2):544-51. doi: 10.1111/j.1432-1033.1997.00544.x.
The alpha2macroglobulin-receptor-associated protein (RAP) binds to the alpha2macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known alpha2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands. In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415], to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively. Structural analysis by 1H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of alpha-helices, whereas domain 3 as a separate domain appears only to be marginally stable. Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I-RAP and 125I-alpha2M* (methylamine-activated alpha2M) for binding to alpha2MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.
α2巨球蛋白受体相关蛋白（RAP）可与α2巨球蛋白受体/低密度脂蛋白受体相关蛋白（α2MR/LRP）结合，后者是一种多功能细胞表面受体，已知可结合并内化多种大分子配体。研究表明，RAP可抑制所有已知的α2MR/LRP配体的结合。突变研究表明，RAP的不同部分特异性参与了多种配体结合的抑制作用。在本文中，我们提供了实验证据，可将先前[Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415]提到的RAP中三重内部序列相似性元素，分别归属于三个结构域，即结构域1、2和3，它们分别包含RAP的第18 - 112、113 - 218和219 - 323位残基。通过1H-NMR光谱进行的结构分析表明，结构域1和2作为独立结构域具有相似的二级结构，几乎完全由α螺旋组成，而结构域3作为独立结构域似乎仅具有微弱的稳定性。针对125I-RAP和125I-α2M*（甲胺激活的α2M）与α2MR/LRP结合的重组RAP结构域1、2和3以及双结构域1 + 2和2 + 3的配体竞争滴定表明：（a）单个结构域中的功能完整性在很大程度上得以保留；（b）抑制测试配体的重要决定因素位于结构域1和3的C末端区域。
