Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Biochem J. 2020 Sep 18;477(17):3199-3217. doi: 10.1042/BCJ20200228.
Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin-GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.
肝表达抗菌肽 2（LEAP2）最近被鉴定为胃肽 ghrelin 的 G 蛋白偶联受体 GHSR1a 的竞争性拮抗剂。LEAP2 通过调节 ghrelin-GHSR1a 系统在能量代谢中发挥重要作用。然而，LEAP2 与 GHSR1a 结合的分子机制在很大程度上尚不清楚。在本研究中，我们首先对人 LEAP2 的 N 端片段进行丙氨酸扫描诱变，证明带正电荷的 Arg6 和芳香性 Phe4 对于 LEAP2 与 GHSR1a 的结合是必需的。为了鉴定与 LEAP2 的必需 Arg6 和 Phe4 相互作用的受体残基，我们对 GHSR1a 进行了广泛的定点突变。在人 GHSR1a 的细胞外区域的所有保守负电荷残基被突变后，只有突变的 Asp99 导致 GHSR1a 与 LEAP2 的结合比与 ghrelin 的结合受到更大的损害，这表明 GHSR1a 的绝对保守的 Asp99 可能与 LEAP2 的必需 Arg6 相互作用。在人 GHSR1a 的预测配体结合口袋中的五个保守 Phe 残基被突变后，其中三个被鉴定为对 GHSR1a 与 LEAP2 的结合很重要。根据 GHSR1a 的结构模型，我们推断相邻的 Phe279 和 Phe312 可能与 LEAP2 的必需 Phe4 相互作用，而 Phe119 可能与 LEAP2 的芳香性 Trp5 相互作用。本研究为 LEAP2 与其受体的相互作用提供了新的见解，并将有助于未来研究中为 GHSR1a 设计新型配体。
