The isolated <i>Stachel</i> peptide of the adhesion G protein-coupled receptor GPR126 is intrinsically disordered

Several members of the adhesion subfamily of G protein-coupled receptors (aGPCRs) are capable of self-activation by an internal agonist sequence (aka the <i>Stachel</i>) that's exposed upon removal or conformational changes of the N-terminal fragment of the receptor. Synthetic peptides derived from the <i>Stachel</i> sequence can be used as exogenous agonists. In the inactive form, the <i>Stachel</i> is sequestered as the β13-strand within the 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 <i>Stachel</i> AlphaFold3 and Pepfold4 models predicted with high confidence for the entire aGPCR subfamily, disorder predictions and biophysical experiments reveal a predominantly disordered conformation in solution. Investigating the ADGRG6/GPR126 <i>Stachel</i> 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/GPR126 <i>Stachel</i> peptide is primarily disordered, but small populations may adopt a helix-containing conformer that seems to support a conformational-selection activation mechanism.