BPS2026 – Does the Stachel really need to sting? Computationally investigating tethered agonist-dependent activation of aGPCRs

Although more than a third of approved drugs target G-protein-coupled receptors (GPCRs), one subclass remains pharmacologically untouched—adhesion GPCRs (aGPCRs). Members of this subfamily are expressed widely and have been implicated in a variety of human ailments including multiple cancers, male infertility, and chronic inflammation disorders. A recent explosion in the experimental structures of aGPCRs have revealed a unique subfamily-wide architecture characterized by a soluble N-terminal autoproteolysis-inducing (GAIN) domain and variable number of extracellular ligand interaction domains (ECDs).The cleavage site is adjacent to a short stretch of mostly hydrophobic residues known as the Stachel or tethered agonist (TA) that remains attached to the seven transmembrane domain and for more than half of aGPCRs is necessary for activation. One of the major models for aGPCR activation involves complete release of the TA from the GAIN domain induced by ligand binding to the ECDs or mechanical stimulation and subsequent binding to the 7TM cleft. However, this model fails to account for aGPCR-mediated signaling in the absence of cleavage, and the mechanistic relationship between TA binding and TA-dependent activation remains unclear. To test this model, we are currently using enhanced sampling molecular dynamics (MD) simulations to predict the full transition between inactive and active states of two aGPCRs from different subgroups. This allows us to assess how changes in the GAIN domain lead to partial or full TA exposure, how TA binding correlates with protein activation and how well this mechanism holds for different family members. We plan to relate the protein behavior we observe in our MD to information gleaned from HDX-MS experiments. Together, this will provide a high-resolution picture of how aGPCR activation occurs in real time.