Binding between a distal C-terminus fragment of cannabinoid receptor 1 and arrestin-2

Abstract

Internalization of G-protein-coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB1(5P)(454-473)). Complexes formed between the peptide and human arrestin-2 (wt-arr2(1-418)) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2(1-382)) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1(5P)(454-473) adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh(7P)(330-348)), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1(5P)(454-473) peptide binds to tr-arr2(1-382) with higher affinity than to the full-length wt-arr2(1-418). As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred NOE data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB1(5P)(454-473) peptide.

Figure 1

A) SDS PAGE analysis of fractions obtained from heparin column purification of arrestin. The supernatant (S), flow through (FT), washes (W^1-4) and Elutions (E^1-2) are shown. B) Western blot analysis of supernatant (S) and Elution1 (E1) from panel A. C) SDS PAGE analysis of gel filtration purified E1 from panel A. The overloaded lane shows a clean product. D) CD spectrum of wt-arr2_1-418 showing a predominance of beta-sheet and some evidence of alpha-helical character.

Figure 2

Fingerprint and side chain regions of the ¹H NOESY spectra, with 1D projections, recorded at 10°C of A) CB1_454-473, B) CB1^5P_454-473 in 10 mM phosphate buffer, 100 mM NaCl at pH=7.0. The following long range nOes were observed: Thr461:CH₃-Ser463:HN, Thr461:HN-Ser463:HN, Thr461:CH₃-Val464:CH^β, Thr461:CH₃-Val464:HN, Val464:CH₃-Asp467:HN, Ser465:CH^α-Asp467:HN, Thr466:HN-Thr468:HN, Met462:CH^β₂-Thr468:HN

Figure 3

(A) Ensemble of five low energy structures of free CB1^5P_454-473 calculated using long range nOes. These demonstrate a persistent turn in the peptide. B) Stick representation of CB1^5P_454-473 highlighting intramolecular hydrogen bonding that may serve to restrict the flexibility of the peptide in solution.

Figure 4

Titration of the truncated arrestin with CB1^5P_454-473, showing the calorimetric response as successive injections of ligand are added to the reaction cell. Panel B depicts the binding isotherm of the calorimetric titration shown in panel A. The continuous line represents the least-squares fit of the data to a single-site binding model.

Figure 5

2D transferred NOESY spectrum of A) Unbound 1.0 mM CB1^5P_454-473 B) Mixture of 1.0 mM CB1^5P_454-473 with 100 μM human arrestin-2 showing the presence of additional chemical exchange peaks. Arrows indicate short and medium range interactions and circle indicates a long range interaction in this region.

Figure 6

nOe interactions observed for CB1^5P_454-473 bound to A) wild-type arrestin-2 and B) truncated arrestin-2. Long range nOes included Met462:CH^β₂-Thr468:HN and Met462:${\mathrm{CH}}_2^{\gamma }$-Glu471:HN. Ambiguous nOes observed included, Thr454:CH₃-Ser469:CH₂, Val455:CH₃-Asp467:HN Val455:CH₃-Ser469:HN.

Figure 7

A) Ensemble of the six low energy structures of full-length arrestin-2 bound CB1^5P_454-473 with backbone superimposition. B) Lowest energy structure with phosphorylated residues shown as ball and stick C) Ramachandran plot and the structural statistics of bound CB1^5P_454-473.

Figure 8

A) Ribbon representation of CB1^5P_454-473, colored in blue (at the N-terminus) to red (at the C-terminus) binding the wild type arrestin colored in green. B) Ribbon representation of CB1^5P_454-473 shown binding to a surface representation of N-domain binding site highlighted in green. The blue line highlights the placement of the C-terminus residues that occlude the binding cavity. The positively charged residues in the binding site are labeled and colored in red. Interactions with phosphorylated residues Thr460, Ser463 and Ser465 of the peptide are labeled a, b and c respectively. C) Ribbon representation of CB1^5P_454-473, colored in blue (at the N-terminus) to red (at the C-terminus) binding the truncated arrestin colored in green. Note the absence of the C-terminus. D) Ribbon representation of CB1^5P_454-473 shown binding to a surface representation of the N-domain binding site highlighted in green. The positively charged residues in the binding site are labeled and colored in red. Interactions with phosphorylated residues Thr460, Ser463 and Ser465 of the peptide are labeled a, b and c respectively. The list of active and passive residues chosen to define the binding site in HADDOCK is shown in the table.