Intrinsic conformational equilibria position arrestin-2 for activation.

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. Here, we use methyl-specific nuclear magnetic resonance spectroscopy, temperature-dependent chemical shift analysis, and relaxation measurements to characterize the intrinsic conformational landscape of full-length human arrestin-2 in solution. We identify two distinct equilibria with separable thermodynamic and kinetic signatures. A slow, enthalpically-favored process sensed by interdomain isoleucines I241 and I317 populates an active-like, interdomain-twisted conformation at physiological temperatures. In parallel, a faster, globally distributed equilibrium consistent with C-terminal tail release exhibits opposing thermodynamic behavior. Dynamic analyses reveal localized rigidification in the active-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 intrinsically samples activation-relevant conformations in the absence of binding partners, providing a solution-state framework for arrestin pre-activation and signaling competence.