Alternative extracellular disulfide bond formation by N-terminal cysteines supports functional expression of odorant...

Mammalian odorant receptors (ORs), a large family of G protein-coupled receptors, rely on conserved intramolecular disulfide bonds for proper folding and their function. However, some ORs lack the typical cysteine in extracellular loop 2, raising questions regarding alternative structural-stabilization mechanisms. Here, we identified and characterized an unconventional disulfide bond between a unique N-terminal cysteine and extracellular loop 2 cysteine in OR51E2, the first mammalian OR with an experimentally resolved structure. Using mutagenesis and functional assays in heterologous cells, we demonstrated that this N-terminal cysteine is essential for receptor surface expression and ligand responsiveness. Furthermore, AlphaFold structural predictions correlated with experimental data, indicating that alternative disulfide bonds contribute to receptor stability. These findings reveal a flexible disulfide bonding strategy in ORs, suggesting that alternative extracellular cysteine linkages can compensate for missing conserved bonds, thereby supporting receptor folding and functional diversification within the G protein-coupled receptor family.