Abstract
Binding mechanism of arrestin requires photoactivation and phosphorylation of the receptor protein rhodopsin, where the receptor bound phosphate groups cause displacement of the long C-tail ‘activating’ arrestin. Mutation of arginine 175 to glutamic acid (R175E), a central residue in the polar core and previously predicted as the ‘phosphosensor’ leads to a pre-active arrestin that is able to terminate phototransduction by binding to non-phosphorylated, light-activated rhodopsin. Here, we report the first crystal structure of a R175E mutant arrestin at 2.7 Å resolution that reveals significant differences compared to the basal state reported in full-length arrestin structures. These differences comprise disruption of hydrogen bond network in the polar core, and three-element interaction including disordering of several residues in the receptor-binding finger loop and the C-terminus (residues 361–404). Additionally, R175E structure shows a 7.5° rotation of the amino and carboxy-terminal domains relative to each other. Consistent to the biochemical data, our structure suggests an important role of R29 in the initial activation step of C-tail release. Comparison of the crystal structures of basal arrestin and R175E mutant provide insights into the mechanism of arrestin activation, where binding of the receptor likely induces structural changes mimicked as in R175E.
Highlights
N-domain in an extended conformation, closely interacting with the two elements and ending on the top of the polar core[8,9,10,11,12]
The mutant arrestin residue 175 from arginine to glutamic acid (R175E) was expressed in Saccharomyces cerevisae F11 α strain and purified as described in methods
R175E crystallized in space group P212121, with one molecule per asymmetric unit
Summary
It is likely that in the pre-activated state the loop becomes highly flexible ‘searching’ for the correct binding site in the receptor, which is stabilized once bound to the receptor as seen in the rhodopsin-arrestin crystal structure where it adopts a α -helical conformation[28]. Previous mutagenesis studies have shown that like R175E, mutation of residues in the polar core, C-tail and those involved in the three-element interaction result in pre-active arrestins that bind light-activated rhodopsin irrespective of its phosphorylation state[3]. For the highly conserved ion pair R175 and D296, it was proposed that neutralization or charge reversal of the residues result in mutants that mimic the effect of phosphate groups from the receptor, allowing them to bypass the need of receptor phosphorylation[3] Formation of this state would involve structural rearrangements in the polar core, C-tail and three-element interaction. Additional structural changes are expected to take place for optimal binding of the receptor such as seen in the loop conformations in case of rhodopsin-arrestin[28] and β -arrestin phosphopeptide complexes[15]
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