title: Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the κ opioid receptor using a soluble protein scaffold.
abstract: The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the κ opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble β-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.
authors.forename: Johannes
authors.initials: J
authors.lastname: Björnerås
authors.forename: Martin
authors.initials: M
authors.lastname: Kurnik
authors.forename: Mikael
authors.initials: M
authors.lastname: Oliveberg
authors.forename: Astrid
authors.initials: A
authors.lastname: Gräslund
authors.forename: Lena
authors.initials: L
authors.lastname: Mäler
authors.forename: Jens
authors.initials: J
authors.lastname: Danielsson
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004399:Dynorphins / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005240:Feasibility Studies; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017473:Receptors, Opioid, kappa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D008024:Ligands; D050505:Mutant Proteins; C515653:OPRK1 protein, human; D010446:Peptide Fragments; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D004399:Dynorphins; D013482:Superoxide Dismutase