Intrinsic conformational equilibria position arrestin-2 for activation

Abstract

Arrestins regulate G protein‐coupled receptor (GPCR) signaling by undergoing large‐scale conformational rearrangements, yet the solution‐state equilibria that underlie arrestin pre‐activation remain poorly defined. While prior studies identified slow conformational exchange at the interdomain interface, these minor states could not be structurally linked to activation because their resonances broaden beyond detection upon receptor binding. Here, we use multinuclear NMR spectroscopy to characterize the intrinsic conformational landscape of full‐length, human arrestin‐2 in solution. We identify two distinct, pre‐existing conformational equilibria that mirror key steps of the receptor‐driven activation process. First, a slow exchange process populates a receptor‐bound‐like, interdomain‐twisted conformation at physiological temperatures. In parallel, a faster, globally distributed equilibrium populates a state consistent with C‐terminal tail release. Dynamic analyses reveal localized rigidification in the receptor‐bound‐like minor states despite arrestin's overall flexibility, while backbone relaxation data indicate widespread μs–ms conformational exchange. Together, these results demonstrate that arrestin‐2 acts as a preorganized scaffold that intrinsically samples receptor‐binding relevant conformations in the absence of binding partners. This provides a solution‐state framework for arrestin pre‐activation and establishes a dynamic fingerprint for future ligand‐dependent studies.