Toward an Understanding of Agonist Binding to HumanOrexin-1 and Orexin-2 Receptors with G-Protein-CoupledReceptor Modeling and Site-Directed Mutagenesis

摘要

The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep–wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a moleculardynamics simulation and to help distinguish between different GPCRsubstates. The results demonstrate how these newly developed methodsof structural assessment for GPCRs can be used to provide a workingmodel of neuropeptide–Orexin receptor interaction.