A Dynamic Picture of the Early Events in Nociceptin Binding to the NOP Receptor by Metadynamics

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.