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Production, crystallization and preliminary X‐ray diffraction of the Gαs α‐helical domain in complex with a nanobody
Author(s) -
Triest Sarah,
Wohlkönig Alexandre,
Pardon Els,
Steyaert Jan
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14020962
Subject(s) - g protein coupled receptor , crystallography , crystallization , chemistry , epitope , biophysics , receptor , biology , biochemistry , antibody , genetics , organic chemistry
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 P 2 1 , 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.