Binding of β-arrestins to phosphorylated GPCRs is thought to involve two types of interaction between a receptor and a β-arrestin molecule6
. A phosphate sensor engages the phosphorylated carboxy terminus or third intracellular loop of the receptor, and a conformational sensor recognizes the agonist-induced, active conformation of the core of the receptor (). Using mass spectrometry-based conformational mapping, we have previously used a V2
vasopressin receptor-derived phosphopeptide (V2
Rpp) to investigate activation of β-arrestins1 and 25,7
. Binding to V2
Rpp recapitulates functionalities of receptor activated β-arrestins, such as enhanced clathrin binding5
. Thus, we reasoned that crystallographic study of a complex of β-arrestin1 with V2
Rpp would provide insight into the mechanisms of receptor-mediated β-arrestin activation. However, well-ordered crystals of β-arrestin1 bound to V2
Rpp could not be obtained. This is presumably due to the significant conformational flexibility of activated arrestin molecules, as was recently determined for visual arrestin by NMR spectroscopy8
. Given the success of antigen binding fragments (Fabs)9
in stabilizing particular GPCR conformations, we sought to identify and characterize conformationally-selective Fabs that stabilize the V2
Rpp bound, active conformation of β-arrestin1.
Fab30 specifically recognizes and stabilizes an active state of β-arrestin1
We utilized a minimalist synthetic Fab phage display library11
to select several high affinity Fabs that selectively recognize the β-arrestin1:V2
Rpp complex (Fig. S1
). One of these, Fab30, displays striking selectivity for the activated conformation of β-arrestin1 induced by V2
Rpp (). In order to ensure that Fab30 stabilizes a physiologically relevant conformation of β-arrestin1, we investigated whether this Fab could facilitate interaction between a receptor and β-arrestin1. Here, we used the previously described chimeric receptor β2
R which has an identical carboxy terminus to V2
Rpp, and which also has unaltered ligand binding characteristics compared to the wild-type β2
adrenergic receptor (β2
. Complexes of GPCRs with either G proteins or β-arrestins display an enhanced affinity for agonists due to the allosteric interactions among the agonist, the receptor and the transducer (G protein or β-arrestin)13,14
. Addition of exogenous β-arrestin1 to the membranes containing phosphorylated β2
R resulted in a small fraction of the receptor in high agonist affinity state compared to receptor alone (). Addition of Fab30 significantly increased the percentage of receptors in the high affinity state. Furthermore, a direct physical stabilization of the receptor:β-arrestin1 complex by Fab30 was revealed by co-immunoprecipitation (). Here, we present a 2.6 Å crystal structure of the β-arrestin1:V2
Rpp:Fab30 complex ().
The overall structure of activated β-arrestin1 exhibits a wide variety of pronounced structural changes compared to previously determined inactive state structures. Most notably, the N- and C-domains of β-arrestin1 undergo a substantial twist relative to one another (), with a 20° rotation around a central axis. The V2Rpp binds to the N-domain at a similar location to the β-arrestin1 carboxy terminus in inactive structures and makes extensive contacts, primarily through charge-charge interactions of V2Rpp phosphates with β-arrestin1 arginine and lysine side chains (compare with , ).
Conformational changes associated with β-arrestin1 activation
V2Rpp interactions with β-arrestin1
This binding mode is consistent with previous limited proteolysis studies that revealed protection of the N-domain of β-arrestin1 in the presence of V2
. Additionally, crosslinking experiments on the β-arrestin1:V2
Rpp complex in the absence of Fab30 show that the amino terminus of the V2
Rpp is in close proximity to K77, consistent with our structure (Fig. S2
). Like the β-arrestin1 carboxy terminus, V2
Rpp binds β-arrestin1 by extending the N-domain β-sandwich fold. Unlike the carboxy terminus, however, V2
Rpp binds as an anti-parallel β-strand. This binding mode may serve as a general mechanism by which arrestins recognize the phosphorylated loops and carboxy-terminal tails of receptors.
In addition to the large interdomain rearrangement, the N-domain and central loops show large structural changes associated with β-arrestin1 activation. Several loops have been implicated in various aspects of β-arrestin activation and receptor interaction15
. These include the “finger loop” (residues 63-75), the “middle loop” (residues 129-140) and the “lariat loop” (residues 274-300). Each of these loops exhibits activation-dependent conformational changes (). Comparison of these loops with inactive structures of β-arrestin1 shows the considerable flexibility in each loop in the inactive conformation, but a more marked change in conformation upon β-arrestin activation (). The crystal structure reveals that the V2
Rpp occludes the inactive conformation of the finger loop, which has been shown to be important for arrestin discrimination between active and inactive GPCRs16
Rpp may stabilize an extended conformation of this loop to facilitate contact with the receptor core (). It is noteworthy that the finger and middle loops above are not at the β-arrestin1:Fab30 interaction interface (Fig. S3
), and therefore, the conformational reorientation observed for these loops most likely reflects activation-dependent changes in β-arrestin1. However, finger loop residues 63-67 and lariat loop residues 285-287 engage in crystal lattice contacts (Fig. S4
), so some caution is warranted in the interpretation of conformational changes in these regions.
Two major sets of intramolecular interactions have been proposed to constrain arrestins in an inactive conformation: the three-element interaction and the polar-core interaction. The three element interaction consists of interactions between β-strand I, α-helix I and the carboxy terminus of arrestin17
. Disruption of this interaction by mutagenesis yields arrestins that are partially phosphorylation-independent in their binding to receptor17
, suggesting a key role for this interaction network in recognizing phosphorylated receptors. The crystal structure of β-arrestin1 shows that two well-conserved residues, K10 and K11 on β-strand I, make charge-charge interactions with phosphorylated residues pS363 and pS357 of V2
Rpp (). Indeed, mutagenesis of the corresponding lysines in visual arrestin significantly decreases binding to phosphorylated, active rhodopsin, suggesting that these residues serve as essential phosphate recognition elements17
. Furthermore, previous limited proteolysis studies have indicated that the carboxy terminus of both visual and β-arrestins is released upon activation as part of the disruption of the three element interaction5,18,19
. Consistent with this model, we observe that the β-arrestin1 carboxy terminus is displaced by the V2
Rpp (). The β-arrestin1 carboxy terminus contains a clathrin binding site that has been previously characterized to be important for GPCR internalization20
. Hence, displacement of the carboxy terminus upon phosphopeptide binding and β-arrestin1 activation is likely an important contributor to clathrin-mediated GPCR internalization. In comparison to previous models, however, binding of V2
Rpp and displacement of the carboxy terminus does not dramatically alter the secondary structure of β-strand I. While we observe abundant charge-charge interactions between β-arrestin1 and V2
Rpp, it is noteworthy that neither the specific sequence of the phosphorylation sites nor the net number of phosphates is conserved among various receptors. Therefore, it remains to be seen how β-arrestins fine-tune their interaction with such a large number of receptors.
The second constraint that stabilizes the inactive conformation of arrestins is the polar core18
, consisting of five interacting charged residues: D26, R169, D290, D297, and R393. Disruption of the polar core by mutagenesis yields phosphorylation-independent mutants of both visual arrestin and β-arrestin117,21
. Charge reversal of R169 or D290 in β-arrestin1 (R175 and D290 in visual arrestin) disrupts this interaction network, yielding arrestins that can bind non-phosphorylated, activated receptors. Based on these studies, R169 was previously proposed to be a critical phosphate sensor in β-arrestin1, and disruption of the polar core was proposed to be required for β-arrestin1 activation21
. Contrary to this model, R169 does not make any direct contacts with V2
Rpp phosphates, suggesting that direct interaction between R169 and receptor phosphates is not required for arrestin activation (). However, binding of V2
Rpp does disrupt the polar core. V2
Rpp binding to β-arrestin1 displaces the arrestin carboxy terminus, and in doing so, removes R393 from the polar core. Residues D290 and D297 also lose interactions within the polar core, and this is accompanied by a dramatic twisting of the lariat loop, which contains both D290 and D297. Therefore, it is possible that the disruption of the polar core is driven by the excess negative charge in this region following displacement of the arrestin carboxy terminus residue R393. Interestingly, the side chain of K294, a residue within the lariat loop, flips toward the N-domain upon activation and engages pT360. It is possible that K294 recognition of phosphates provides an additional driving force for lariat loop rearrangement, and may therefore stabilize β-arrestin1 in an active conformation. This observation in the crystal structure is consistent with crosslinking experiments, which reveal the disappearance of an intra-peptide crosslink between K292 and K294 in the presence of V2
Rpp (Fig. S5
), indicating that V2
Rpp induces a conformation like that seen in the crystal structure even in the absence of Fab30.
While domain rearrangement upon arrestin activation has been previously proposed, the observed 20° twisting of the N- and C- domains of β-arrestin1 upon activation is unanticipated. Biochemical studies have shown that sequential deletion of the visual arrestin hinge region connecting the N- and C- domains results in a progressive decrease in the ability of arrestin to bind phosphorylated, light-activated rhodopsin. This suggests a requirement for relative movement of the two domains for efficient interaction with activated receptors22
. However, the twisting motion observed here stands in contrast to the “clamshell” hypothesis advanced previously23
. Considering the large number of interaction partners of β-arrestins during cellular signaling24
, it is tempting to speculate that the twisting movement of the two domains upon arrestin activation may expose interaction interfaces with such binding partners.
Recent NMR and double electron-electron resonance (DEER) studies have assessed the conformational changes induced in visual arrestin upon interaction with phosphorylated, light-activated rhodopsin8,25
. Intriguingly, NMR spectroscopy of activated visual arrestin revealed significant line broadening attributed to intermediate timescale conformational dynamics over the entire arrestin molecule8
. Within such an ensemble of activated arrestin conformations, Fab30 likely stabilizes a conformation of β-arrestin1 that preferentially binds activated GPCRs. Furthermore, distance restraints for activated visual arrestin derived from DEER experiments are highly consistent with the active structure of β-arrestin1 presented here (Fig. S6
). Most notably, the large conformational change observed for the middle loop by DEER spectroscopy upon binding light-activated, phosphorylated rhodopsin is also evident in the crystal structure of activated β-arrestin1. Given the importance of this region in maintaining the inactive conformation of visual arrestin, the agreement in conformational changes within arrestin suggests that the V2
Rpp bound, active conformation of β-arrestin1 presented here represents a similar state to that of arrestin in complex with a phosphorylated, activated GPCR. This further suggests that the conformational changes associated with activation and receptor binding are conserved throughout the arrestin family. However, the binding stoichiometry between GPCRs and arrestins still remains to be fully established. Recent biochemical studies have suggested that two rhodopsin molecules may simultaneously bind one arrestin26
. The extensive and specific contacts between V2
Rpp and the β-arrestin1 N-domain likely preclude another receptor carboxy terminus from binding β-arrestin1. However, it is possible that an arrestin molecule bound to the phosphorylated carboxy terminus of a receptor could interact with the 7TM core of another receptor. Additional data, including a crystal structure of a GPCR:β-arrestin complex, will be required to clarify this.
In summary, we present here the first structure of an activated arrestin bound to the phosphorylated carboxy terminus of a GPCR. The structure not only provides the atomic details of a potentially general GPCR-β-arrestin interaction interface, but also offers novel insights into the activation process of arrestins, and reveals a large interdomain twisting associated with activation. These findings will facilitate future efforts to understand the structural basis for β-arrestin activation and signaling. Such studies may ultimately yield insight into how GPCRs achieve such a large breadth of signaling complexity.