|Title||Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.|
|Publication Type||Journal Article|
|Year of Publication||2014|
|Authors||Triest, S., A. Wohlkonig, E. Pardon, and J. Steyaert|
|Journal||Acta Crystallogr F Struct Biol Commun|
|Date Published||2014 Nov|
|Keywords||Animals, Cattle, Crystallization, GTP-Binding Protein alpha Subunits, Gs, Humans, Protein Binding, Single-Domain Antibodies, X-Ray Diffraction|
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.
|Alternate Journal||Acta Crystallogr F Struct Biol Commun|
|PubMed Central ID||PMC4231852|
Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.