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NMR analysis of dynamics and interactions of two G protein-coupled receptors : the HIV-1 coreceptor CCR5 and the β1-adrenergic receptor

Grahl, Anne. NMR analysis of dynamics and interactions of two G protein-coupled receptors : the HIV-1 coreceptor CCR5 and the β1-adrenergic receptor. 2018, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_13551

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Abstract

This thesis aims to provide insight into structural and dynamic features of the pharmaceutical highly relevant membrane protein family of G protein-coupled receptors (GPCRs) by Nuclear Magnetic Resonance (NMR) spectroscopy. A quantitative description of the backbone dynamics of the turkey β1-adrenergic receptor (β1AR) was achieved by the analysis of NMR relaxation data. For the chemokine receptor and HIV-1 coreceptor CCR5 ligand interactions were studied by solution as well as solid-state NMR.
Chapter 1 provides an introduction to the potential and challenges of NMR for membrane protein analysis. The biological function of GPCRs and the current structural and dynamic knowledge are discussed with a particular focus on the turkey β1AR. Furthermore, the chapter describes the role of the chemokine receptor CCR5 as major coreceptor for HIV-1 infection and the potential of chemokine analogs as HIV-1 entry inhibitors and signaling modulators of CCR5.
Chapter 2 gives an overview of the relaxation phenomena observed in solution NMR to study protein dynamics. The theoretical basis for extracting dynamic information in the nano- to millisecond time range from experimental relaxation rates and conformational exchange is presented.
Chapter 3 presents a detailed picture of the global and local dynamics of the thermostabilized turkey β1AR obtained from experimental 1H and 15N NMR relaxation. Local backbone motions at 14 different sites of the β1AR in its apo form, six binary ligand complexes ranging from inverse agonists to agonists and a ternary agonist/G protein mimicking nanobody complex were investigated. Allosteric coupling across the β1-adrenergic receptor was revealed by the pivoting of transmembrane helix 6 on the extracellular side upon intracellular effector site binding. A manuscript with parts of these results is currently under revision (Grahl et al., NMR backbone dynamics reveals mechanism of ligand to effector site allosteric coupling in the β1‑adrenergic receptor). The final section of the chapter reports on the favorable effect of partial deuteration on the transverse relaxation and spectral appearance of β1AR.
Chapter 4 describes the successful expression of human, engineered CCR5 in insect cells and its characterization in different detergent micelles to yield a stable receptor sample for structural characterization. Insect cell expression schemes were implemented to obtain uniform or amino acid-specific isotope-labeled material for backbone NMR studies. CCR5 was globally characterized by 15N relaxation rates indicating no significant nanosecond motions of the receptor independent from the detergent micelle, but exchange dynamics in the micro- to millisecond time range. Initial NMR data on CCR5 have been published (Franke, Opitz, Isogai, Grahl et al., Production of isotope-labeled proteins in insect cells for NMR. J. Biomol. NMR, 2018). In addition, promising initial cryo-electron microscopy images of CCR5 have been obtained, which may allow a future full analysis.
Chapter 5 describes the characterization of the potent anti-HIV-1 entry inhibitor 5P12‑RANTES-E66S at physiological pH. Interactions studies were conducted with detergent micelles and heparin disaccharide as a model for the binding to the glycosaminoglycan layer on the cell surface.
Chapter 6 provides insight into the complex of CCR5 with the chemokine analog 5P12‑RANTES-E66S. A stable, monomeric complex was obtained, and initially characterized by solution NMR. An extended binding interface for CCR5 could be mapped on isotope-labeled 5P12-RANTES-E66S and micro- to millisecond dynamics of the complex-bound chemokine analog could be detected. High-quality solid-state NMR spectra of the immobilized chemokine•receptor complex were obtained promising for future detailed characterization.
Chapter 7 describes the initial characterization of wild type-like human β2-adrenergic receptor by backbone NMR experiments.
Advisors:Grzesiek, Stephan and Schertler, Gebhard
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
UniBasel Contributors:Grahl, Anne and Grzesiek, Stephan
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13551
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (xiv, 176 Seiten)
Language:English
Identification Number:
Last Modified:30 Sep 2020 04:30
Deposited On:16 Jun 2020 13:25

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