The isolated Stachel peptide of the adhesion G protein-coupled receptor ADGRG6 is predominantly disordered with local helical propensity.

Several members of the adhesion subfamily of G protein-coupled receptors (aGPCRs) are capable of self-activation by an internal agonist sequence (i.e., the Stachel) that is exposed upon removal or conformational changes of the N-terminal fragment of the receptor. Synthetic peptides derived from the Stachel sequence can be used as exogenous agonists. In the inactive form of the full-length receptor, the Stachel is sequestered as the β13 strand within the G protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain, but it engages the seven transmembrane region as a helix when it is either an intramolecular sequence or a synthetic peptide. Little is known about the molecular details underlying this transition, but we hypothesize that a disordered conformation is central to this intermediate state in receptor activation. Despite the primarily helical Stachel AlphaFold3 and PEP-FOLD4 models predicted with high confidence for the entire aGPCR subfamily, computational predictions and biophysical experiments reveal a predominantly disordered conformation in solution. Investigating the ADGRG6 (also known as GPR126) Stachel peptide, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments reveal a predominantly random-coil conformation in aqueous buffer, polar detergent micelles, and zwitterionic lipids. Titration of trifluoroethanol uncovered a two-state equilibrium between an unfolded and helix-containing conformation with NMR localizing a single-turn helix to residues L846-L849. Taken together, these data indicate the ADGRG6 Stachel peptide is primarily disordered with a subset adopting partial helical structures, likely requiring the steric hindrance of the receptor binding pocket to fully induce helix formation in an induced-fit mechanism.