Mechanistic insights into adhesion GPCR autoproteolysis by a multiscale computational approach

In cis -autoproteolysis, proteins self-catalyze cleavage of their peptide chains. In the GAIN domain of adhesion G protein-coupled receptors, cis -autoproteolysis yields a tethered agonist critical for receptor activation. Contrary to current reaction models, the GAIN HL|T catalytic triad is insufficient for cleavage. Here, we investigated the cleavage mechanism in the rat ADGRL1 GAIN domain using multiscale modeling combining molecular dynamics and QM/MM simulations to investigate cis -autoproteolysis as a once-in-a-lifetime event. We present an updated and unique GAIN domain cis -autoproteolysis mechanism. The initial N-O acyl shift proceeds via a hydroxy-oxazolidine intermediate, sterically shifting subsequent reaction steps away from the triad base H ^GPS.-2 . Water on its opposing side, coordinated by acidic residue E656 ^H6.50 , is essential for completing the N-O acyl shift facilitating ester hydrolysis. Our study provides detailed mechanistic insights into cis -autoproteolysis, highlighting how conserved residues and structured water networks adjacent to the triad enable a chemically precise reaction within an inherently flexible protein not evolutionarily optimized for high catalytic rate. These findings deepen our understanding of aGPCR processing and may apply broadly to other autoproteolytic proteins, offering a framework to explore similarly elusive one-time cleavage events in biology.