Struktur und Dynamik der GPCR-vermittelten Aktivierung von G-Proteinen mit Lösungs-NMR
Biochemie
Zusammenfassung der Projektergebnisse
In this project, we investigated the structure and interactions of an inhibitory G-protein with partner proteins using structural methods, such as NMR and cryo-EM. We first performed NMR resonance assignments of Ga in complex with GDP and studied the complex formation with the nucleotide exchange factor and Ga chaperone Ric8A using NMR chemical shift perturbation experiments. We found that Ric8A interacts with the C-terminal a-helix5 of Ga, which is also the canonical binding site of Ga for a GPCR. This binding site was later confirmed by crystallography by our collaborator Prof. Stephen Sprang. Next, we established an NMR assay utilizing chemical labeling with 13C-MMTS at cysteine side chains in a GPCR. Such a conformational assay was necessary since we used stabilized versions of neurotensin receptor obtained by directed evolution for our structural investigations. This procedure led to the accumulation of an increasing number of point mutations to enhance receptor stability and expression level in E. coli. Our NMR assay provided a reliable readout for receptor switching functionality which enabled us to use this approach to guide backmutation experiments to restore the receptor functionality even in the presence of multiple mutations. It turned out that single back-mutations are sufficient to restore functionality, offering the advantage of high receptor stability and high expression level in E. coli. In the last part of this project, together with our collaborators Prof. Gerhard Wagner and Prof. Andreas Plückthun, we used cryo-EM to determine the structure of a stabilized neurotensin receptor in complex with a heterotrimeric G-protein in lipid nanodiscs without the need to use nanobodies for complex stabilization. The use of negatively charged lipids was essential to obtain a high-affinity complex that is suitable for high-resolution structure determination without further reagents. The obtained structural information provided novel information on the mechanistic details of the nucleotide exchange activity of a GPCR when bound to a G-protein.
Projektbezogene Publikationen (Auswahl)
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NMR backbone and methyl resonance assignments of an inhibitory G-alpha subunit in complex with GDP. Biomolecular NMR Assignments, 13(1), 131-137.
Goricanec, David & Hagn, Franz
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Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A. Structure, 27(7), 1137-1147.e5.
Zeng, Baisen; Mou, Tung-Chung; Doukov, Tzanko I.; Steiner, Andrea; Yu, Wenxi; Papasergi-Scott, Makaia; Tall, Gregory G.; Hagn, Franz & Sprang, Stephen R.
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Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling. ChemBioChem, 22(1), 139-146.
Goba, Inguna; Goricanec, David; Schum, Dominik; Hillenbrand, Matthias; Plückthun, Andreas & Hagn, Franz
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Cryo-EM structure of an activated GPCR–G protein complex in lipid nanodiscs. Nature Structural & Molecular Biology, 28(3), 258-267.
Zhang, Meng; Gui, Miao; Wang, Zi-Fu; Gorgulla, Christoph; Yu, James J.; Wu, Hao; Sun, Zhen-yu J.; Klenk, Christoph; Merklinger, Lisa; Morstein, Lena; Hagn, Franz; Plückthun, Andreas; Brown, Alan; Nasr, Mahmoud L. & Wagner, Gerhard
