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Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-2713-7731
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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
Abstract [en]

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.
Keyword [en]
G protein coupled receptor, membrane proteins, neuropeptide, NMR, protein chimeras
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-105256DOI: 10.1111/febs.12626ISI: 000336732600014OAI: oai:DiVA.org:su-105256DiVA: diva2:729081
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:6;

Available from: 2014-06-25 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Dynorphin A – Interactions with receptors and the membrane bilayer
Open this publication in new window or tab >>Dynorphin A – Interactions with receptors and the membrane bilayer
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 42 p.
National Category
Biophysics
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-92769 (URN)
Presentation
2013-09-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 15:00 (English)
Opponent
Supervisors
Available from: 2013-08-20 Created: 2013-08-20 Last updated: 2014-09-29Bibliographically approved
2. The opioid peptide dynorphin A: Biophysical studies of peptide–receptor and peptide–membrane interactions
Open this publication in new window or tab >>The opioid peptide dynorphin A: Biophysical studies of peptide–receptor and peptide–membrane interactions
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA.

The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event.

With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge.

Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 72 p.
Keyword
dynorphin A, opioid receptor, neuropeptide, bicelles, NMR, diffusion, decomposition
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-107766 (URN)978-91-7649-011-2 (ISBN)
Public defence
2014-10-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

Available from: 2014-10-09 Created: 2014-09-29 Last updated: 2014-10-13Bibliographically approved

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