Structurome-wide analysis reveals intracellular loop 2 folding as an activation-associated feature of class A G protein-coupled receptors.

Activation of class A G protein-coupled receptors (GPCRs) is commonly described through rearrangement of the transmembrane-bundle and engagement of the Gα α5 helix, whereas the conformational behavior of intracellular loop 2 (ICL2) has not been examined systematically across the structural record. Here, I classified ICL2 as folded, unfolded, or disordered in 1358 experimental class A GPCR structures using a backbone-dihedral criterion and evaluated the classification against activation state, transducer class, crystallization fusions, ICL2-proximal modulators, and resolution. The assignment was additionally assessed using Dictionary of Secondary-Structure of Proteins and a literature-anchored benchmark set. In canonical fusion-free structures, ICL2 was folded in 84% of active structures compared with 53% of inactive structures [odds ratio, 4.74; two-sided Fisher exact test; descriptive P = 7 × 10-13]. The association remained evident after controlling for receptor overrepresentation, within receptors represented in both states, and among active structures without an annotated transducer. Crystallization fusions were associated with increased apparent ICL2 folding in inactive structures. Folded and unfolded ICL2 also showed distinct Gα contact patterns, while sequence features displayed only modest associations with folding. A geometrically compatible contact between ICL2 position 34.53 and the conserved D3.49 residue accompanied the folded state but was not universal. These exploratory structural analyses identify ICL2 folding as an activation-associated feature of class A GPCRs without establishing a causal mechanism or a determinant of transducer selectivity. SIGNIFICANCE STATEMENT: This structure-wide analysis shows that intracellular loop 2 (ICL2) folding is strongly enriched in active compared with inactive class A GPCR structures, identifying ICL2 as an intracellular structural readout of receptor activation. The association is retained across major structural controls, while transducer analyses show that ICL2 folding is most prevalent in Gs-bound complexes but does not by itself define coupling specificity.