From the Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada.
the Department of Oral Biology, University of Buffalo, Buffalo, New York 14214, and.
J Biol Chem. 2018 Nov 9;293(45):17523-17535. doi: 10.1074/jbc.RA118.004489. Epub 2018 Sep 25.
Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG-heparan sulfate (HS) interactions, and formation of the OPG-HS-receptor activator of nuclear factor κB ligand (RANKL) ternary complex. Employing hydrogen-deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure-function relationship of the full-length OPG protein.
先前对骨保护素（OPG）的结构研究主要局限于其 N 端配体结合结构域，OPG 是骨重塑和破骨细胞生成的关键负调控因子。现在已知，OPG 的三个 C 端结构域在其功能中也起着至关重要的作用，通过介导 OPG 二聚体形成、OPG-硫酸乙酰肝素（HS）相互作用以及 OPG-HS-核因子 κB 受体激活剂配体（RANKL）三元复合物的形成。我们采用氘氢交换 MS 方法，在此研究了全长 OPG 与 HS 或 RANKL 在溶液中形成的复合物的结构。我们的数据揭示了 OPG 结构的两个显著方面。首先，我们发现 N 端和 C 端结构域之间的连接比以前认为的要牢固得多，这可能是由于第四富含半胱氨酸结构域和第一个死亡结构域之间的疏水相互作用。其次，我们观察到位于两个独立 C 端结构域中的两个疏水区簇直接有助于 OPG 二聚体的形成，可能通过形成疏水二聚体界面。通过定点突变，我们进一步证明完整的二聚体界面对于 OPG 的生物学活性至关重要。我们的研究代表了朝着解析全长 OPG 蛋白结构-功能关系迈出的重要一步。
