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.
N-methyl-d-aspartate receptors are ionotropic glutamate receptors that mediate the slow component of excitatory neurotransmission. Modulators of NMDA receptor function have long been evaluated as pharmacotherapies to treat neurological disorders such as Parkinson’s Disease, Alzheimer’s disease, schizophrenia, stroke, and other neuropsychiatric and neurodegenerative disorders. We describe the mechanisms of action and explore the structural determinants underlying a phenomenon where GluN2B-specific negative allosteric modulators from the 93- and 96-series can be inverted to act as positive allosteric modulators by mutations at two GluN1 residues in their binding pocket located within the amino terminal domain. Mutations at GluN1-Y109 that invert the activity could each conceivably strengthen the interaction between the residue and 93-31, resulting in a switch from enhancement to relief of tonic inhibition of the receptor by protons. The demonstration that this binding site is capable of being tuned for either inhibition or potentiation provides an opportunity to design novel positive allosteric modulators for potential therapeutic development in humans.Significance StatementMutations of two residues in the NMDA receptor GluN1 subunit amino terminal domain independently interconvert GluN2B-selective negative allosteric modulators into positive allosteric modulators. This work provides a path for the discovery of the structural requirements for GluN2B-specific positive allosteric modulation.
The mechanosensitive Piezo1 channel is crucial for the regulation of calcium (Ca2+) signaling within myeloid tissue macrophages. However, the current lack of comprehensive pharmacological characterization for existing Piezo1 agonists complicates the assessment of Piezo1 agonism as a viable therapeutic strategy. We report inhibition of the voltage-gated Kv1.3 potassium (K+) channel as a critical off-target effect of the most widely used Piezo1 agonists, Yoda1 and Yoda2. Kv1.3 is essential for the activation and innate immune functions of tissue macrophages. Patch-clamp electrophysiological experiments demonstrate that Yoda1 (IC50 = 9.0 μM) and Yoda2 (IC50 = 5.9 μM) state-independently inhibit Kv1.3 by accelerating channel inactivation. Functionally, blockade of Kv1.3 by Yoda2 in Piezo1-deficient bone marrow-derived macrophages depolarizes membrane potential and reduces store-operated calcium entry and interleukin-1β release. Using RosettaLigand docking and molecular dynamics simulations, we identified a network of key residues near the Kv1.3 outer pore as the potential binding site. Our results suggest that Kv1.3 blockade likely contributes substantially to the reported anti-inflammatory effects of Yoda1 and Yoda2. This provides a plausible mechanism that aligns with the established principle that reducing Ca2+ signaling is anti-inflammatory, while helping to reconcile existing literature discrepancies regarding Piezo1 activation. SIGNIFICANCE STATEMENT: Targeting Ca2+ channels such as Piezo1 is vital for controlling chronic inflammation, but selectivity is challenging. This study reveals that the best-known Piezo1 agonists exhibit a critical off-target effect by blocking the Kv1.3 channel, which is essential for macrophage and microglial immune function. This concurrent inhibition reduces inflammatory Ca2+ signaling and interleukin-1β secretion. This work underscores the necessity for rigorous selectivity screening of Piezo1 drugs and establishes Kv1.3 inactivation modification as a viable strategy for designing novel anti-inflammatory therapeutics.
