Conformational dynamics of the active state of β-arrestin 1.

β-arrestins (βarr) regulate the signaling and trafficking of G protein-coupled receptors (GPCRs) in numerous physiological processes and have been implicated in various diseases. Structural and kinetic insights into how ligand-mediated GPCR activation drives βarr coupling and activation remain limited, with the binding mechanism of the phosphorylated GPCR C-terminal tails, such as that of the vasopressin receptor-2 (V2Rpp), and the conformation of the entire βarr tail in the active state still unknown. Here, we simulated both the basal and V2Rpp-bound states of βarr1 with temperature replica-exchange molecular dynamics (TREMD) simulations to probe the activation mechanism of βarr1. Compared to conventional MD, our TREMD simulations, employing an unprecedented 200-replica setup, significantly broadened conformational sampling while preserving the basal state. Our analysis showed that, without the bound Fab30 antibody fragment, the main body of V2Rpp-bound βarr1 tended to transition toward the basal conformation; however, binding of V2Rpp in the N-domain groove allosterically oriented the finger loop to point upward for core engagement with a GPCR. Furthermore, V2Rpp dissociation events suggest that its binding involves a sliding movement along the N-domain groove, during which its phosphorylated residues p3 and p4 transiently occupy the S5 site to facilitate repositioning of p5 into the S5 site, thereby triggering a zippering process of p1 to p3. The dynamic 62-residue βarr1 tail explored a vast conformational space, forming transient secondary structures, and could favorably anchor on the main body's back side and within the central crest crevice. These findings elucidate key mechanistic steps underlying βarr1 activation.