Production, crystallization and preliminary X-ray diffraction of the G?s ?-helical domain in complex with a nanobody.

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 G?s ?-helical domain (AHD). To stabilize GPCR-G-protein complexes in a nucleotide-free form, nanobodies were selected that target the flexible G?sAHD. One of these nanobodies, CA9177, was co-crystallized with the G?sAHD. 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 P2?, with unit-cell parameters a=44.07, b=52.55, c=52.66?Å, ?=90.00, ?=107.89, ?=90.00°. The structure of this specific nanobody reveals its binding epitope on G?sAHD and will help to determine whether this nanobody could be used as crystallization chaperone for GPCR-G-protein complexes.

SUBMITTER: Triest S 

PROVIDER: S-EPMC4231852 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature