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- Title
Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.
- Authors
Triest, Sarah; Wohlkönig, Alexandre; Pardon, Els; Steyaert, Jan
- Abstract
GPCR-G-protein complexes are one of the most important components of cell-signalling cascades. Extracellular signals are sensed by membrane-associated G-protein-coupled receptors (GPCRs) and transduced via G proteins towards intracellular effector molecules. Structural studies of these transient complexes are crucial to understand the molecular details of these interactions. Although a nucleotide-free GPCR-G-protein complex is stable, it is not an ideal sample for crystallization owing to the intrinsic mobility of the Gαs α-helical domain (AHD). To stabilize GPCR-G-protein complexes in a nucleotide-free form, nanobodies were selected that target the flexible GαsAHD. One of these nanobodies, CA9177, was co-crystallized with the GαsAHD. Initial crystals were obtained using the sitting-drop method in a sparse-matrix screen and further optimized. The crystals diffracted to 1.59 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 44.07, b = 52.55, c = 52.66 Å, α = 90.00, β = 107.89, γ = 90.00°. The structure of this specific nanobody reveals its binding epitope on GαsAHD and will help to determine whether this nanobody could be used as crystallization chaperone for GPCR-G-protein complexes.
- Subjects
G proteins; EXTRACELLULAR signal-regulated kinases; GUANINE nucleotide exchange factors; MOLECULAR chaperones; ANTIGEN processing
- Publication
Acta Crystallographica: Section F, Structural Biology Communications, 2014, Vol 70, Issue 11, p1504
- ISSN
2053-230X
- Publication type
Article
- DOI
10.1107/S2053230X14020962