The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin

Abstract

Signaling of the prototypical G protein coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin-arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/ peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C-terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, GαCT binding and both ArrFL-1 binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.Unlike other class A GPCRs the photoreceptor rhodopsin contains the light-sensitive cofactor retinal as a ligand, which is covalently bound to the opsin apoprotein. Upon light-induced cis/trans isomerization of retinal and subsequent alterations within the binding pocket, the signal propagates via GPCR-conserved interhelical networks towards the cytoplasmic surface of the receptor, where major structural rearrangements take place. The most prominent structural change is the rotational outward tilt of transmembrane (TM) helix 6, which opens a cytoplasmic binding crevice and thus constitutes the main prerequisite for binding and activation of signaling via the G protein transducin (1, 2). Even though first identified in rhodopsin, for many other GPCRs homologous structural changes have been shown to exist, and a common structural and functional framework for GPCRs is being developed (3).

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations

“…NMR data on the m-opioid receptor shows G-biased ligands favor more open intracellular conformations, while agonist activation of a b-arrestin-biased mutant promotes more closed conformations (Okude et al, 2015). It further accords with work demonstrating the visual arrestin finger loop, the key interaction motif, binds two distinct conformations of active rhodopsin (Elgeti et al, 2018). Since b-arrestin can bind to GPCRs through both a higher affinity interaction with the GRK-phosphorylated carboxy terminus and a lower affinity interaction with the TM core (Latorraca et al, 2018;Shukla et al, 2014), the enforced proximity of b-arrestin resulting from the carboxy terminal interaction could facilitate its binding to occluded conformations or to very low populations of open conformations.…”

Structural Insights into β-arrestin/CB1 Receptor Interaction: NMR and CD Studies on Model Peptides

“…In agreement with our observations, various studies have Our structural studies demonstrated that the β-arr1 [63][64][65][66][67][68][69][70][71][72][73][74][75][76] peptide also formed helical conformations in DPC and TFE (Figure 5). In agreement with our observations, various studies have indicated that in its activated state, the β-arrestin finger loop adopts helical conformations [55,56,62]. However, it is important to note that conformational plasticity of the finger loop was observed in previously reported GPCR/arrestin complexes [51][52][53][54][55].…”

“…indicated that in its activated state, the β-arrestin finger loop adopts helical conformations [55,56,62]. However, it is important to note that conformational plasticity of the finger loop was observed in previously reported GPCR/arrestin complexes [51][52][53][54][55].…”

DEER Analysis of GPCR Conformational Heterogeneity

“…The DEER distance distributions indicate residual structural heterogeneity, especially for the rhodopsin–arrestin-1 complex. This finding suggests that either the receptor or the transducer exist in more than one conformation, a hint at different architectures of this complex with potentially distinct function [ 31 , 93 ]. DEER, due to its superior applicability to large, structural heterogeneous proteins and protein complexes, represents a predestined experimental method to track down those alternative “flavors” [ 94 ].…”