Expression, purification, and preliminary cryo-EM analysis of the human urotensin 2 receptor/urotensin 2 complex

Human urotensin 2 (hU2) interacts with the urotensin 2 receptor (UR2R), a member of the G protein-coupled receptor (GPCR) family. UR2R signaling is known to induce vasoconstriction and promote cell proliferation, and its overexpression is strongly associated with cardiovascular diseases. Despite continued efforts to develop UR2R antagonists, structural information on hU2 binding to UR2R remains unrevealed for developing highly effective therapeutic candidates. Here, we present a high-yield expression and purification workflow to enable cryo-electron microscopy (cryo-EM) studies of active UR2R/hU2 complex. Full-length and truncated UR2R constructs were cloned and expressed in Spodoptera frugiperda 9 (Sf9) insect cells. Following hU2 binding, UR2R was purified using Talon resin and a reverse Ni-NTA column, with an intermediate detergent exchange from n-dodecyl-β-D-maltoside (DDM) to lauryl maltose neopentyl glycol (LMNG) to improve sample homogeneity. Final polishing by size-exclusion chromatography (SEC) yielded highly purified UR2R/hU2 complex. Using this strategy, we successfully assembled the UR2R^11-336/hU2-mGqi-scFv16 complex, and complex formation was confirmed by cryo-EM 2D classification and SDS page. These optimized methods of UR2R expression, purification, and complex assembly enable to determine high-resolution cryo-EM structure of active UR2R/hU2. Our data will support structure-guided drug discovery targeting UR2R-related diseases.