Crystal structure of pre-activated arrestin p44

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Pharmacology

Background:

• Arrestins regulate G-protein-coupled receptor (GPCR) signaling by blocking G protein interaction and mediating G-protein-independent pathways.
• Arrestin activation involves the displacement of its carboxy-terminal tail (C-tail) by receptor-attached phosphates, enabling binding to active GPCRs.
• While inactive arrestin structures are known, the mechanism of C-tail displacement-induced activation remains unclear.

Purpose of the Study:

• To elucidate the structural mechanism of arrestin activation by determining the crystal structure of a pre-activated arrestin.
• To investigate the role of C-tail displacement in releasing critical receptor-binding loops and facilitating arrestin-GPCR interaction.
• To understand the conformational changes that enable arrestin to bind active GPCRs.

Main Methods:

• X-ray crystallography (3.0 Å resolution) of the bovine arrestin-1 splice variant p44, a C-tail truncated, pre-activated form.
• Site-directed fluorescence spectroscopy to verify conformational alterations and their role in arrestin activation and receptor binding.
• Comparative structural analysis between the pre-activated p44 and basal arrestin-1 states.

Main Results:

• The crystal structure of p44 reveals a conformation profoundly different from basal arrestin-1, characterized by the breakage of the central polar core and interlobe hydrogen bonds.
• A significant rotation (~21°) between the two arrestin lobes was observed, alongside rearrangements in key receptor-binding loops (finger loop, loop 139, and gate loop).
• These conformational changes were validated as critical for arrestin activation and receptor binding, indicating a mechanism where C-tail displacement liberates these loops.

Conclusions:

• Arrestin activation involves C-tail displacement, which releases critical central-crest loops from a restricted to an extended, receptor-interacting conformation.
• Increased flexibility between the arrestin lobes facilitates optimal fitting to the active GPCR surface.
• The findings provide a structural snapshot of an arrestin poised for receptor binding and offer insights into the function of naturally truncated arrestins in the visual system.