International Journal of Biomedicine.2019;9 Suppl_1:S19-S19.
Originally published June 29, 2019
Background: G-protein coupled receptors (GPCR) are drug targets of great current interest, being meanwhile a challenging object for structural research.
Methods and Results: The protein of interest needs to be expressed in necessary amounts. To obtain folded and functionally active receptor, usually eukaryotic systems are used, and the protein is located on the cytoplasmic membrane (Lv et al. 2016). To deliver a GPCR to the cell surface signaling peptides and their combinations are used (Shepard et al. 2013), however successful combination varies for each GPCR in a particular cell type.
A GPCR must be stable in the membrane-mimetic systems like micelles or nanodiscs, as well as lipidic cubic phase in case of X-ray crystallography. To stabilize a GPCR, fusion partners are put on the protein N-terminus or into the 3rd intracellular loop (ICL-3) (Chun et al. 2012). This helps to fix a receptor in one conformational state, as well as helps to form crystal contacts. ICL-3 insertions are inapplicable for the complexes with G-proteins that are popular for the Cryo-EM structures. Several software tools are used, that help to predict stabilizing point mutations and fusion partner position (Popov et al. 2018; Pándy-Szekeres et al. 2018).
To enhance particular GPCR parameters, like expression, stability or diffraction resolution, N- or C-terminal fragments as well as transmembrane region parts are borrowed from those receptors that are known to be on the top of those parameters, the nearest homologues are preferred. Thus A1 adenosine receptor was crystallized with the parts from muscarinic 4 and adenosine A2A receptors (Glukhova et al. 2017), and for the better β2AR-arrestin binding GPCR C-terminal region was replaced for the one from AVPR2 (Shukla et al. 2014).
Conclusions: GPCR construct engineering is an inherent tool for structural biology, it requires deep preliminary analysis, hard searching effort, and a bit of luck.