期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 108, 期 38, 页码 16086-16091出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1113645108
关键词
G protein-coupled receptor; negative stain electron microscopy; random conical tilt
资金
- Lundbeck Foundation Junior Group
- Fund for Scientific Research of Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen)
- Institute for the encouragement of Scientific Research and Innovation of Brussels
- National Institute of General Medical Sciences (NIGMS) [GM008270, R01-GM083118, R01-GM068603]
- National Institute of Neural Disorders and Stroke [R01-NS28471]
- Mather Charitable Foundation
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [R01-DK090165]
- National Institutes of Health [AI076961, AI08192, AI2008031, CA118595, GM20501, GM066170, GM093325, RR029388]
- Michigan Diabetes Research and Training Center, NIDDK [P60DK-20572]
- University of Michigan
- Lundbeck Foundation [R37-2009-3457] Funding Source: researchfish
The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied beta(2)-adrenoceptor (beta(2)AR) in complex with the heterotrimeric G protein Gs (G alpha s beta gamma). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the alpha-helical domain of G alpha s appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the alpha-helical domain on the Ras-like domain of G alpha s. Molecular modeling of the alpha-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of G alpha s-GTP gamma S. These data argue that the alpha-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.
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