Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.
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.
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Elsene, Belgium.
Sarah
S
Triest
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Elsene, Belgium.
Alexandre
A
Wohlkönig
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Elsene, Belgium.
Els
E
Pardon
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Elsene, Belgium.
Jan
J
Steyaert
D000818:Animals; D002417:Cattle; D003460:Crystallization; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction
D061905:Single-Domain Antibodies; D019205:GTP-Binding Protein alpha Subunits, Gs
G protein; GPCR transmembrane signalling; nanobody-enabled X-ray crystallography