Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold
2014 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 3, 814-824 p.Article in journal (Refereed) Published
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 kappa 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 beta-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.
Place, publisher, year, edition, pages
2014. Vol. 281, no 3, 814-824 p.
G protein coupled receptor, membrane proteins, neuropeptide, NMR, protein chimeras
Research subject Biophysics
IdentifiersURN: urn:nbn:se:su:diva-105256DOI: 10.1111/febs.12626ISI: 000336732600014OAI: oai:DiVA.org:su-105256DiVA: diva2:729081
FunderSwedish Research CouncilKnut and Alice Wallenberg Foundation