Della Longa Stefano, Arcovito Alessandro
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Rome, Italy.
Biophys J. 2016 Sep 20;111(6):1203-1213. doi: 10.1016/j.bpj.2016.07.004.
Nociceptin (NCC, also known as FQ (N/OFQ)) is the 17-amino acid neuropeptide, endogenous ligand for the G-protein-coupled receptor (NOP, also known as ORL-1). In this study, starting from the recently reported x-ray structure at pH 7 of NOP in complex with an antagonist, new insights, to our knowledge, on the binding geometry of NCC to NOP have been provided in silico. After a rigid docking of NCC in an α-helix conformation, molecular dynamics (MD) and metadynamics (METAD), a method for the analysis of free-energy surfaces (FES), were performed on the protein-peptide complex. Free-energy profiles were obtained as a function of the α-helix content of different segments of the 17-mer ligand, and a structural ensemble of conformations of NCC, corresponding to the minimum of the FES, was extracted, thus representing the NCC bound to the inactive form of NOP. The structural features were compared with many known experimental data. The pose of the "message" domain (residues 1-4) of NCC differs from that of the known NOP antagonists, as being slightly slipped deeper inside the protein core. A residual α-helix content in the central part of the peptide (residues 4-9) is maintained, whereas the C-terminal segment (residues 13-17) is unstructured and highly flexible. An important stabilization due to interactions with residues D130 and D110 of the receptor has been found, in agreement with the large decrease in agonist potency reported for the D130A and D110A mutants. The importance of the extracellular domain 2 (ECL2) in the selectivity toward the endogenous ligand has been confirmed. A pivotal role for the conserved residue N133 is suggested and further supported by a study of the N133A in silico mutant. Accordingly, N133 can work as a molecular microswitch driving the change between the inactive and active NOP conformations, in the framework of an extended H-bond and water network rearrangement in the deep binding site.
痛敏肽（NCC，也称为FQ (N/OFQ)）是一种由17个氨基酸组成的神经肽，是G蛋白偶联受体（NOP，也称为ORL-1）的内源性配体。在本研究中，基于最近报道的NOP与拮抗剂在pH 7条件下的X射线结构，我们通过计算机模拟提供了关于NCC与NOP结合几何结构的新见解。在将处于α-螺旋构象的NCC进行刚性对接后，对蛋白质-肽复合物进行了分子动力学（MD）和元动力学（METAD，一种用于分析自由能表面（FES）的方法）。获得了作为17聚体配体不同片段α-螺旋含量函数的自由能分布，并提取了对应于FES最小值的NCC构象结构集合，从而代表与无活性形式NOP结合的NCC。将这些结构特征与许多已知的实验数据进行了比较。NCC“信息”结构域（第1-4位残基）的构象与已知的NOP拮抗剂不同，它稍微更深地滑入蛋白质核心内部。肽段中部（第4-9位残基）保留了残余的α-螺旋含量，而C端片段（第13-17位残基）无结构且高度灵活。已发现与受体的D130和D110残基相互作用导致重要的稳定性，这与D130A和D110A突变体激动剂效力大幅降低的报道一致。已证实细胞外结构域2（ECL2）在内源性配体选择性中的重要性。通过对计算机模拟的N133A突变体的研究，表明保守残基N133起关键作用，并得到进一步支持。因此，在深层结合位点扩展的氢键和水网络重排框架内，N133可作为驱动NOP无活性和活性构象之间变化的分子微开关。
