Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a
Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a
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鉴定 LEAP2 与受体 GHSR1a 结合的关键残基和关键相互作用
DOI：
10.1042/bcj20200228
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发表时间：
2020-09-01
影响因子：
4.1
通讯作者：
Guo, Zhan-Yun
中科院分区：
文献类型：
--
作者：
Li, Hao-Zheng;Shou, Li-Li;Guo, Zhan-Yun
肝脏表达抗菌肽2(Leap2)是胃肽Ghrelin的同源受体--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-末端片段进行了丙氨酸扫描突变，证明了带正电荷的Arg6和芳香族Phe4是Leap2与GHSR1a结合所必需的。为了确定与Leap2必需的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胞外区所有保守的负电荷残基都发生突变后，只有Asp99突变对与Leap2结合的GHSR1a造成的损害比与ghrelin结合的损害更大，这表明GHSR1a的绝对保守的Asp99可能与Leap2的必需Arg6相互作用。在预测的人GHSR1a配体结合口袋中的五个保守的Phe残基发生突变后，其中三个残基被鉴定为对GHSR1a与Leap2的结合具有重要作用。根据GHSR1a的结构模型，我们推测邻近的Phe279和Phe312可能与Leap2的基本Phe4相互作用，而Phe119可能与Leap2的芳香族Trp5相互作用。本研究对Leap2与其受体的相互作用提供了新的见解，并将有助于在未来的研究中设计新型的GHSR1a配体。
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.