uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Mutagenesis and Computational Modeling of Human G‑Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Show others and affiliations
2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, 7987-7998 p.Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor–agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013. Vol. 52, no 45, 7987-7998 p.
National Category
Natural Sciences Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-154994DOI: 10.1021/bi400830cISI: 000330017700012OAI: oai:DiVA.org:uu-154994DiVA: diva2:432635
Available from: 2011-08-04 Created: 2011-06-14 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
Open this publication in new window or tab >>Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs.

The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes.

This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity.

A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246.

In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 687
Keyword
Y2-receptor, NPY, PYY, G-protein-coupled receptor, mutagenesis, receptor binding, zebrafish, evolution
National Category
Pharmacology and Toxicology
Research subject
Pharmacology
Identifiers
urn:nbn:se:uu:diva-156635 (URN)978-91-554-8117-9 (ISBN)
Public defence
2011-09-21, B:41, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-08-31 Created: 2011-08-04 Last updated: 2018-01-12Bibliographically approved
2. Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
Open this publication in new window or tab >>Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.

We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target.  In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.

Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1105
Keyword
computer simulations, molecular dynamics, ligand binding, free energy perturbation, linear interaction energy, binding free energy, homology modelling, structure prediction, alanine scanning, site-directed mutagenesis, hERG, GPCR, neuropeptide Y, HIV-1 reverse transcriptase, integron integrase
National Category
Theoretical Chemistry Structural Biology Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-212103 (URN)978-91-554-8823-9 (ISBN)
Public defence
2014-01-31, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-01-10 Created: 2013-12-05 Last updated: 2014-01-24
3. Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
Open this publication in new window or tab >>Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions.

The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization.

A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals.

Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours.

In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.

Place, publisher, year, edition, pages
Uppsala, Sweden: Acta Universitatis Upsaliensis, 2014. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1040
Keyword
Neuropeptide Y, genome duplication, Evolution, vertebrate, Pharmacology, Modelling, Kinetics
National Category
Evolutionary Biology Pharmacology and Toxicology Cell and Molecular Biology Neurosciences Biochemistry and Molecular Biology Cell Biology Structural Biology
Research subject
Bioinformatics; Biology with specialization in Evolutionary Genetics; Biology with specialization in Evolutionary Functional Genomics; Pharmaceutical Pharmacology
Identifiers
urn:nbn:se:uu:diva-233461 (URN)978-91-554-9059-1 (ISBN)
Public defence
2014-11-21, C2, 305, Husargatan 3, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-10-31 Created: 2014-10-06 Last updated: 2018-01-11

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Xu, BoFällmar, HelenaBoukharta, LarsPruner, JasnaLundell, IngridGutiérrez-de-Terán, HugoÅqvist, JohanLarhammar, Dan

Search in DiVA

By author/editor
Xu, BoFällmar, HelenaBoukharta, LarsPruner, JasnaLundell, IngridGutiérrez-de-Terán, HugoÅqvist, JohanLarhammar, Dan
By organisation
PharmacologyComputational and Systems Biology
In the same journal
Biochemistry
Natural SciencesMedical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 450 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf