Florey Institute of Neuroscience and Mental Health, University of Melbourne Parkville, VIC, Australia ; Department of Biochemistry and Molecular Biology, University of Melbourne Parkville, VIC, Australia.
Front Endocrinol (Lausanne). 2013 Feb 22;4:13. doi: 10.3389/fendo.2013.00013. eCollection 2013.
Relaxin-3 is a highly conserved neuropeptide in vertebrate species and binds to the Class A G protein-coupled receptor (GPCR) RXFP3. Relaxin-3 is involved in a wide range of behaviors, including feeding, stress responses, arousal, and cognitive processes and therefore targeting of RXFP3 may be relevant for a range of neurological diseases. Structural knowledge of RXFP3 and its interaction with relaxin-3 would both increase our understanding of ligand recognition in GPCRs that respond to protein ligands and enable acceleration of the design of drug leads. In this study we have used comparative sequence analysis, molecular modeling and receptor mutagenesis to investigate the binding site of the native ligand human relaxin-3 (H3 relaxin) on the human RXFP3 receptor. Previous structure function studies have demonstrated that arginine residues in the H3 relaxin B-chain are critical for binding interactions with the receptor extracellular loops and/or N-terminal domain. Hence we have concentrated on determining the ligand interacting sites in these domains and have focused on glutamic (E) and aspartic acid (D) residues in these regions that may form electrostatic interactions with these critical arginine residues. Conserved D/E residues identified from vertebrate species multiple sequence alignments were mutated to Ala in human RXFP3 to test the effect of loss of amino acid side chain on receptor binding using a Eu-labeled relaxin-3 agonist. Finally data from mutagenesis experiments have been used in ligand docking simulations to a homology model of human RXFP3 based on the peptide-bound chemokine receptor 4 (CXCR4) structure. These studies have resulted in a model of the relaxin-3 interaction with RXFP3 which will inform further interrogation of the agonist binding site.
松弛素-3 是脊椎动物中高度保守的神经肽，与 A 类 G 蛋白偶联受体 (GPCR) RXFP3 结合。松弛素-3 参与广泛的行为，包括进食、应激反应、觉醒和认知过程，因此靶向 RXFP3 可能与一系列神经疾病有关。RXFP3 的结构知识及其与松弛素-3 的相互作用都将增加我们对响应蛋白质配体的 GPCR 中配体识别的理解，并能够加速药物先导物的设计。在这项研究中，我们使用比较序列分析、分子建模和受体突变来研究天然配体人松弛素-3（H3 松弛素）在人 RXFP3 受体上的结合位点。先前的结构功能研究表明，H3 松弛素 B 链中的精氨酸残基对于与受体细胞外环和/或 N 端结构域的结合相互作用至关重要。因此，我们专注于确定这些结构域中的配体相互作用位点，并集中研究这些区域中的谷氨酸 (E) 和天冬氨酸 (D) 残基，这些残基可能与这些关键精氨酸残基形成静电相互作用。从脊椎动物物种的多重序列比对中鉴定出保守的 D/E 残基，并在人 RXFP3 中突变为 Ala，以使用 Eu 标记的松弛素-3 激动剂测试氨基酸侧链缺失对受体结合的影响。最后，突变实验的数据用于配体对接模拟，以基于肽结合趋化因子受体 4 (CXCR4) 结构的人 RXFP3 同源模型。这些研究产生了松弛素-3 与 RXFP3 相互作用的模型，这将为进一步研究激动剂结合位点提供信息。
