Understanding the Differential Selectivity of Arrestins toward the Phosphorylation State of the Receptor

Abstract

Proteins in the arrestin family exhibit a conserved structural fold that nevertheless allows for significant differences in their selectivity for G-protein coupled receptors (GPCRs) and their phosphorylation states. To reveal the mechanism of activation that prepares arrestin for selective interaction with GPCRs, and to understand the basis for these differences, we used unbiased molecular dynamics simulations to compare the structural and dynamic properties of wild type Arr1 (Arr1-WT), Arr3 (Arr3-WT), and a constitutively active Arr1 mutant, Arr1-R175E, characterized by a perturbation of the phosphate recognition region called "polar core". We find that in our simulations the mutant evolves toward a conformation that resembles the known preactivated structures of an Arr1 splice-variant, and the structurally similar phosphopeptide-bound Arr2-WT, while this does not happen for Arr1-WT. Hence, we propose an activation allosteric mechanism connecting the perturbation of the polar core to a global conformational change, including the relative reorientation of N- and C-domains, and the emergence of electrostatic properties of putative binding surfaces. The underlying local structural changes are interpreted as markers of the evolution of an arrestin structure toward an active-like conformation. Similar activation related changes occur in Arr3-WT in the absence of any perturbation of the polar core, suggesting that this system could spontaneously visit preactivated states in solution. This hypothesis is proposed to explain the lower selectivity of Arr3 toward nonphosphorylated receptors. Moreover, by elucidating the allosteric mechanism underlying activation, we identify functionally critical regions on arrestin structure that can be targeted with drugs or chemical tools for functional modulation.

Molecular mechanism of GPCR-mediated arrestin activation

“…We performed simulations of arrestin-2 (β-arrestin-1) under several conditions, and the results are consistent with a similar activation mechanism (Extended Data Fig. 9), despite certain functional differences between arrestins 21 . A wide variety of previously published data also supports the hypothesis that the receptor core and R P tail each independently promote activation of β-arrestins (Extended Data Table 1).…”

Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification

“…Finally, a short 25-residue arrestin-3-derived peptide lacking most of receptor-binding elements effectively activated JNK3 in vitro and in cells (23). Thus, arrestin-3 can exist in an active (at least in terms of JNK3 activation) conformation without GPCR binding, likely due to its high flexibility revealed by structural work (48) and molecular dynamics modeling (49).…”

The single nucleotideβ-arrestin2 variant, A248T, resembles dynamical properties of activated arrestin

“…All the analyses of the MD trajectories were carried out after removing the first 50 ns, when a relatively stable plateau was reached in backbone root-mean-square-deviations (RMSD). The rotation analysis was made as explained in detail in [14].…”

“…Moreover, the "gate loop" (residues 295-306) [10,11], the "C-loop"(residues 244-249), the "short-helix" (residues 313-317), and the "aromatic core" have been shown to undergo conformational rearrangement upon activation as evident from comparison of crystal structures of inactive and active arrestin [12,13]. These regions have been also proposed to be involved in the activation mechanism [14] (Figure 1), while the "finger loop" (residues 66-75) is primarily required for binding to the receptor [11,15]. As such, any mutation occurring in these regions might prevent either activation of arrestin or receptor binding.…”