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.

中文翻译：

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确定关键残基和关键相互作用，使LEAP2与受体GHSR1a结合。

肝表达的抗菌肽2（LEAP2）最近被确定为G蛋白偶联受体GHSR1a（胃肽ghrelin的同源受体）的竞争性拮抗剂。LEAP2通过调节ghrelin–GHSR1a系统在能量代谢中起重要作用。但是，LEAP2与GHSR1a结合的分子机制尚不清楚。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诱变，并证明带正电荷的Arg6和芳香族Phe4对于LEAP2与GHSR1a的结合至关重要。为了确定与LEAP2的必需Arg6和Phe4相互作用的受体残基，我们对GHSR1a进行了广泛的定点诱变。在人类GHSR1a细胞外区域中所有保守的带负电荷的残基突变后，仅Asp99突变对GHSR1a与LEAP2的结合造成的损害比对与ghrelin的结合的损害大得多，这表明GHSR1a的绝对保守的Asp99可能与LEAP2的必需Arg6相互作用。在人类GHSR1a的预期配体结合口袋中的五个保守的Phe残基突变后，其中三个对GHSR1a与LEAP2的结合很重要。根据GHSR1a的结构模型，我们推论相邻的Phe279和Phe312可能与LEAP2的必需Phe4相互作用，而Phe119可能与LEAP2的芳香族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识，并将有助于在未来的研究中为GHSR1a设计新的配体。这表明GHSR1a的绝对保守的Asp99可能与LEAP2的必需Arg6相互作用。在人类GHSR1a的预期配体结合口袋中五个保守的Phe残基突变后，其中三个对GHSR1a与LEAP2的结合很重要。根据GHSR1a的结构模型，我们推论相邻的Phe279和Phe312可能与LEAP2的必需Phe4相互作用，而Phe119可能与LEAP2的芳香族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识，并将有助于在未来的研究中为GHSR1a设计新的配体。这表明GHSR1a的绝对保守的Asp99可能与LEAP2的必需Arg6相互作用。在人类GHSR1a的预期配体结合口袋中的五个保守的Phe残基突变后，其中三个对GHSR1a与LEAP2的结合很重要。根据GHSR1a的结构模型，我们推论相邻的Phe279和Phe312可能与LEAP2的必需Phe4相互作用，而Phe119可能与LEAP2的芳香族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识，并将有助于在未来的研究中为GHSR1a设计新的配体。其中三个被确定对GHSR1a与LEAP2的结合很重要。根据GHSR1a的结构模型，我们推论相邻的Phe279和Phe312可能与LEAP2的必需Phe4相互作用，而Phe119可能与LEAP2的芳香族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，并将在今后的研究中促进GHSR1a的新型配体的设计。其中三个被确定对GHSR1a与LEAP2的结合很重要。根据GHSR1a的结构模型，我们推论相邻的Phe279和Phe312可能与LEAP2的必需Phe4相互作用，而Phe119可能与LEAP2的芳香族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识，并将有助于在未来的研究中为GHSR1a设计新的配体。