RESUMEN
Orexin 2 receptor (OX2R) is a G protein-coupled receptor (GPCR) whose activation is crucial to regulation of the sleep-wake cycle. Recently, inactive and active state structures were determined from X-ray crystallography and cryo-electron microscopy single particle analysis, and the activation mechanisms have been discussed based on these static data. GPCRs have multiscale intermediate states during activation, and insights into these dynamics and intermediate states may aid the precise control of intracellular signaling by ligands in drug discovery. Molecular dynamics (MD) simulations are used to investigate dynamics induced in response to thermal perturbations, such as structural fluctuations of main and side chains. In this study, we proposed collective motions of the TM domain during activation by performing 30 independent microsecond-scale MD simulations for various OX2R systems and applying relaxation mode analysis. The analysis results suggested that TM3 had a vertical structural movement relative to the membrane surface during activation. In addition, we extracted three characteristic amino acid residues on TM3, i.e., Q1343.32, V1423.40, and R1523.50, which exhibited large conformational fluctuations. We quantitatively evaluated the changes in their equilibrium during activation in relation to the movement of TM3. We also discuss the regulation of ligand binding recognition and intracellular signal selectivity by changes in the equilibrium of Q1343.32 and R1523.50, respectively, according to MD simulations and GPCR database. Additionally, the OX2R-Gi signaling complex is stabilized in the conformation resembling a non-canonical (NC) state, which was previously proposed as an intermediate state during activation of neurotensin 1 receptor. Insights into the dynamics and intermediate states during activation gained from this study may be useful for developing biased agonists for OX2R.
Asunto(s)
Simulación de Dinámica Molecular , Receptores de Orexina , Receptores de Orexina/química , Receptores de Orexina/metabolismo , Transducción de Señal , HumanosRESUMEN
To investigate the dynamics of the orexin 2 receptor, which is a class A G protein-coupled receptor, we recently performed several microsecond-scale molecular dynamics simulations of the wild-type protein, of a mutant that stabilizes the inactive state, and of constitutively active mutants of the class A G protein-coupled receptors. Herein, we review the results of these molecular dynamics simulations of the orexin 2 receptor. In these simulations, characteristic conformational changes were observed in the V3096.40Y mutant. The conformational changes were related to the outward movement of the transmembrane helix 6 and the inward movement of the transmembrane helix 7, which are common structural changes in the activation of G protein-coupled receptors. The index for the quantitative evaluation of the active and inactive states of class A G protein-coupled receptors and the mechanism of the inward movement of the transmembrane helix 7 were examined. In this review, we also discuss the activation mechanism by comparing the structures obtained from the molecular dynamics simulations with the structure of the active state of the orexin 2 receptor clarified by cryo-electron microscopy in the recent years.
RESUMEN
The orexin2 receptor (OX2R), which is classified as a class A G protein-coupled receptor (GPCR), is the target of our study. We performed over 20 several-microsecond-scale molecular dynamics simulations of the wild type and mutants of OX2R to extract the characteristics of the structural changes taking place in the active state. We introduced mutations that exhibited the stable inactive state and the constitutively active state in class A GPCRs. In these simulations, significant characteristic structural changes were observed in the V3096.40Y mutant, which corresponded to a constitutively active mutant. These conformational changes include the outward movement of the transmembrane helix 6 (TM6) and the inward movement of TM7, which are common structural changes in the activation of GPCRs. In addition, we extracted a suitable index for the quantitative evaluation of the active and inactive states of GPCRs, namely, the inter-atomic distance of Cα atoms between x(3.46) and Y(7.53). The structures of the inactive and active states solved by X-ray crystallography and cryo-electron microscopy can be classified using the inter-atomic distance. Furthermore, we clarified that the inward movement of TM7 requires the swapping of M3056.36 on TM6 and L3677.56 on TM7. Finally, we discussed the structural advantages of TM7 inward movement for GPCR activation.