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• W2739933313 abstract "•A rhodopsin-arrestin complex structure with phosphorylated rhodopsin C terminus•Structural mechanism for recognition of phosphorylated rhodopsin by visual arrestin•Phosphorylation codes are a common mechanism of arrestin recruitment by GPCRs G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor’s C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs. G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor’s C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs. G protein-coupled receptors (GPCRs) are a family of membrane receptors that mediate transmembrane signaling through G proteins and arrestins (Pierce et al., 2002Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2100) Google Scholar, Shukla et al., 2011Shukla A.K. Xiao K. Lefkowitz R.J. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling.Trends Biochem. Sci. 2011; 36: 457-469Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). In response to stimuli, GPCRs activate G proteins to regulate the generation of second messengers, which then modulate downstream signaling effectors. To turn off the response, GPCR kinases (GRKs) are recruited to phosphorylate the GPCRs and prepare them for arrestin binding that blocks G protein-mediated signaling and directs the receptors to internalization (Pitcher et al., 1998Pitcher J.A. Freedman N.J. Lefkowitz R.J. G protein-coupled receptor kinases.Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar). Upon binding to GPCRs, arrestins also serve as signaling scaffolds to initiate alternative pathways independent of G proteins. Thus, arrestin binding to receptors is a pivotal event that orchestrates the GPCR signaling network (Gurevich and Gurevich, 2013Gurevich V.V. Gurevich E.V. Structural determinants of arrestin functions.Prog. Mol. Biol. Transl. Sci. 2013; 118: 57-92Crossref PubMed Scopus (57) Google Scholar). Arrestin recruitment by a GPCR is thought to be initiated by the interaction between inactive arrestin and phosphorylated receptor C-tail, which displaces arrestin’s C-tail and converts the inactive arrestin into a pre-activated state for high affinity GPCR binding (Gurevich and Gurevich, 2006bGurevich V.V. Gurevich E.V. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors.Pharmacol. Ther. 2006; 110: 465-502Crossref PubMed Scopus (362) Google Scholar). Our understanding of how arrestins are recruited by activated GPCRs has been enhanced by recent structural studies (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar, Kim et al., 2013Kim Y.J. Hofmann K.P. Ernst O.P. Scheerer P. Choe H.W. Sommer M.E. Crystal structure of pre-activated arrestin p44.Nature. 2013; 497: 142-146Crossref PubMed Scopus (139) Google Scholar, Shukla et al., 2013Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. et al.Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.Nature. 2013; 497: 137-141Crossref PubMed Scopus (321) Google Scholar, Shukla et al., 2014Shukla A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. et al.Visualization of arrestin recruitment by a G-protein-coupled receptor.Nature. 2014; 512: 218-222Crossref PubMed Scopus (365) Google Scholar). Electron microscopy analysis of β2 adrenergic receptor (β2AR) in complex with β-arrestin-1 revealed a dynamic equilibrium of two states: one is the fully-engaged state with arrestin bound to the central cavity of the receptor 7TM helix bundle, and the other is a partially-engaged state with arrestin bound only to the receptor C-terminal tail (Shukla et al., 2014Shukla A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. et al.Visualization of arrestin recruitment by a G-protein-coupled receptor.Nature. 2014; 512: 218-222Crossref PubMed Scopus (365) Google Scholar). The crystal structure of phosphorylated vasopressin-2 receptor (V2R) tail bound to β-arrestin-1 provided a basis for arrestin activation induced by the phosphorylated receptor tail and the conformation of the activated arrestin, which closely resembles the activated state of visual arrestin (Kim et al., 2013Kim Y.J. Hofmann K.P. Ernst O.P. Scheerer P. Choe H.W. Sommer M.E. Crystal structure of pre-activated arrestin p44.Nature. 2013; 497: 142-146Crossref PubMed Scopus (139) Google Scholar, Shukla et al., 2013Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. et al.Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.Nature. 2013; 497: 137-141Crossref PubMed Scopus (321) Google Scholar). A recent XFEL crystal structure of rhodopsin bound to activated visual arrestin revealed a near-atomic resolution interaction of arrestin with the receptor’s 7TM helix bundle (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar). However, the C-terminal tail of rhodopsin was unresolved in that structure. Thus, how arrestin fully engages with a phosphorylated receptor remains an unanswered fundamental question in GPCR signaling. Here, we report a structure of rhodopsin-arrestin complex with additional structural features including the phosphorylated rhodopsin C-terminal tail bound to the N-terminal domain of arrestin. The phosphorhodopsin-arrestin interface displays an extended intermolecular β sheet together with an extensive network of electrostatic interactions formed between positive residues at the N-terminal domain of arrestin and the phosphorylated C-tail of rhodopsin. These structural features were extensively validated by biochemical and biophysical experiments and computer modeling to provide a basis for understanding phosphorylation-mediated arrestin recruitment by GPCRs. The original XFEL crystal structure of rhodopsin bound to arrestin was solved by serial femtosecond crystallography (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar, Zhou et al., 2016Zhou X.E. Gao X. Barty A. Kang Y. He Y. Liu W. Ishchenko A. White T.A. Yefanov O. Han G.W. et al.X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.Sci. Data. 2016; 3: 160021Crossref PubMed Scopus (44) Google Scholar). Using the recently released 0.6.2 version of CrystFEL (White et al., 2016White T.A. Mariani V. Brehm W. Yefanov O. Barty A. Beyerlein K.R. Chervinskii F. Galli L. Gati C. Nakane T. et al.Recent developments in CrystFEL.J. Appl. Cryst. 2016; 49: 680-689Crossref PubMed Scopus (177) Google Scholar), we reprocessed diffraction images of 22,462 micro-crystals collected at the CXI beamline of the Linac Coherent Light Source at SLAC National Laboratory, which yielded 22% more reflections than those used for the original structure. The reprocessed data were of higher quality with better Pearson correlation coefficient (CC∗) and overall signal-to-noise ratio (Tables S1 and S2). The resolution of the new dataset was improved from 3.8 Å to 3.6 Å along the a∗ and b∗ axes and from 3.3 Å to 3.0 Å along the c∗ axis. The crystals were pseudo-merohedrally twinned in P212121 space group with a twin fraction of 0.5 based on L-test analysis (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar). The structure was refined with twin law (k, h, -l) to Rwork and Rfree of 23.4% and 27.2%, respectively, with excellent geometry (Table S3). The overall structure and the newly resolved elements are well supported by electron density maps (Figure S1). The crystal structure contains four T4 lysozyme (T4L)-rhodopsin-arrestin fusion complexes as presented in our previous model (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar), denoted as A, B, C and D, respectively (Figure 1). The four rhodopsin-arrestin complexes in each asymmetric unit adopt nearly identical structures with root-mean-square deviations (RMSDs) of Cα atoms among four complexes between 0.5 and 0.7 Å, in agreement with the previous structure in the modeled regions (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar). Importantly, we observed electron density for the membrane-touching loop of arrestin (C-edge loop) from residues 340–342 (Figure S1C). Molecular dynamics simulation indicated that C-edge loops of arrestins function as membrane anchors, whose engagement with membrane is required for GPCR binding by arrestins (Lally et al., 2017Lally C.C. Bauer B. Selent J. Sommer M.E. C-edge loops of arrestin function as a membrane anchor.Nat. Commun. 2017; 8: 14258Crossref PubMed Scopus (59) Google Scholar). We also observed electron density for poly N-acetyl-D-glucosamine at the N15 glycosylation site of each rhodopsin molecule (Figure S1D) and most of the rhodopsin C-terminal tail (residues 330–343) including two phosphate groups at T336 and S338 (Figures 1, 2, S1E, and S1F). These structural elements were missing in our previous model (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar), indicating that the reprocessed data have improved the structure and revealed additional structural features that are important to rhodopsin-arrestin interaction.Figure 2Interface between Rhodopsin C-Terminal Tail and Arrestin N-Terminal DomainShow full caption(A) The interface between rhodopsin C-tail and arrestin N-terminal domain with the rhodopsin C-tail covered with a 2mFo-DFc density map contoured at 1 σ. Rhodopsin is in green and arrestin in brown.(B) Interface residues between rhodopsin C-tail and arrestin N-terminal domain, with rhodopsin residues colored green and arrestin residues brown.(C) A schematic diagram of the interactions between rhodopsin C-tail residues (green) and arrestin residues (brown). Solid lines and arrows indicate hydrogen bonds and salt bridges, respectively.(D) Charge-distribution surface of the arrestin N-terminal domain in the rhodopsin-arrestin complex with an electrostatic scale from –3 to +3 eV corresponding to red to blue colors. Labeled are the rhodopsin C-tail residues involved in the charge interaction network.See also Figures S1 and S2 and Table S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The interface between rhodopsin C-tail and arrestin N-terminal domain with the rhodopsin C-tail covered with a 2mFo-DFc density map contoured at 1 σ. Rhodopsin is in green and arrestin in brown. (B) Interface residues between rhodopsin C-tail and arrestin N-terminal domain, with rhodopsin residues colored green and arrestin residues brown. (C) A schematic diagram of the interactions between rhodopsin C-tail residues (green) and arrestin residues (brown). Solid lines and arrows indicate hydrogen bonds and salt bridges, respectively. (D) Charge-distribution surface of the arrestin N-terminal domain in the rhodopsin-arrestin complex with an electrostatic scale from –3 to +3 eV corresponding to red to blue colors. Labeled are the rhodopsin C-tail residues involved in the charge interaction network. See also Figures S1 and S2 and Table S3. The most important new feature of the structure is the rhodopsin C-terminal tail that serves as an interface for arrestin recruitment (Figure 2). While all four complexes in the structure are similar, we focus on molecule A because it has the best electron density for phosphorylated T336 and S338, which are critical for arrestin binding (Figures 1 and 2). Of the resolved rhodopsin C-tail, the C-terminal portion from K339 to T342 adopts a β strand that forms an extended intermolecular β sheet with β strand I, from V12 to K16, of the N-terminal domain of arrestin (Figure 2). Residues N-terminal to the C-tail β strand of rhodopsin, D330–S338, form an extended stretch that interacts with arrestin and connects the β strand to helix 8 through a flexible linker region (K325–G329) that is disordered in the crystal structure. Of this region, residues D330–A333 make a turn that is positioned alongside the turn between the β strands I and II of arrestin, with its D330 and E332 electrostatically associated with R19, K167, and K168 of arrestin (Figure 2C). The conformation and position of residues D330–S335 are slightly different among the four complexes in the crystal structure, indicating the dynamic nature of this region. In contrast, the β strand (K339–T342) of the rhodopsin C-tail adopts the same structure in all four complexes (Figures 1C and 1D), indicating its stability and its role as one of the anchor points for rhodopsin-arrestin interaction. While the rhodopsin C-tail contains six serine or threonine residues, mass spectrometry analysis detected phosphate groups at S334, T336, and S338 in our protein samples (Figure S2). In our crystal structure, however, we observed the phosphate group at S338 in all four rhodopsin molecules and at T336 only in molecules A and B. The phosphate group at S334 was not observed in the structure, possibly due to a high disorder in this part of the C-terminal tail (Figure 2). The interaction between rhodopsin and arrestin is primarily maintained by the formation of the intermolecular anti-parallel β sheet between the rhodopsin C-tail β strand and β strand I of arrestin (Figures 1 and 2) and further stabilized by an extensive electrostatic interaction network between the phosphorylated T336 and S338 and the negatively charged residue E341 of rhodopsin and three positively charged pockets on the arrestin surface denoted as A, B, and C (Figure 2D). The three positively charged pockets are formed by three groups of basic residues: K16, R19, and R172 (pocket A), K16, R30, and K301 (pocket B), and K15 and K111 (pocket C), which accommodate phosphate groups at T336 and S338, and the negatively charged E341, respectively, of the rhodopsin C-tail (Figures 2B–2D). In addition, there is another electrostatic interaction interface between negatively charged rhodopsin residues D330, E332, and potentially phosphorylated S334 and a positively charged patch on arrestin formed by R19, K167, and K168 at the tips of the two β sheets of the N-terminal domain of arrestin (Figures 2B and 2D). It is interesting to note that the total number of positive charges on this arrestin N-terminal surface is nine (Figure 2C), which is equivalent to eight or ten total negative charges of the phosphate groups and acidic amino acid residues at the interface (depending on the phosphorylation of S334, detected by mass spectrometry but not observed in our crystal structure), indicating an overall electrostatic balance at this rhodopsin-arrestin interface (Figure 2D). To validate the structure of the C-terminal tail of rhodopsin in the complex, we performed double electron–electron resonance (DEER) experiments, which are widely used to determine the distances between spin-labeled residues in protein complexes (Altenbach et al., 2008Altenbach C. Kusnetzow A.K. Ernst O.P. Hofmann K.P. Hubbell W.L. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation.Proc. Natl. Acad. Sci. USA. 2008; 105: 7439-7444Crossref PubMed Scopus (379) Google Scholar, Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar). We determined the distances from rhodopsin C-tail residues 335, 337, and 342 to arrestin residues 106 or 107, which are located at arrestin helix I. Figure 3 shows the distances from residues 335 and 337 of rhodopsin to residue 107 of arrestin (Figures 3A and 3B) and the distances between residue 342 of rhodopsin and residues 106 and 107 of arrestin (Figures 3C and 3D). These DEER distances are in agreement with those of the structural model, indicating the correct positioning of the C-terminal tail of rhodopsin in the crystal structure. We further validated the rhodopsin C-tail-arrestin interface by site-specific disulfide cross-linking through engineering cysteine pairs across the interface (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar). A total of 13 cysteine mutants of arrestin together with 7 cysteine mutants of rhodopsin were expressed. A total of 63 co-expression combinations were tested and monitored by SDS-PAGE followed by western blotting, and the results are summarized in Figure S3A. Examples show that rhodopsin E332 strongly cross-linked with R19, K167, and K168 of arrestin (Figure 4A), as E332 faces toward these positively charged residues, while D331 of rhodopsin weakly cross-linked, because it faces away from the arrestin surface, with longer distances and unfavorable geometry to form crosslinking with these arrestin residues (Figure 4A). Rhodopsin residues A333, S334, and A335 cross-linked with only R19, and T336 cross-linked with both K16 and R19 of arrestin, indicating that all these residues are in close proximity to R19, while T336 is closer to K16 than A333, S334, and A335 (Figures 4B and 4C). Rhodopsin E341 cross-linked with K111 on helix I, but did not cross-link with residues on β strand I of arrestin, probably due to unfavorable geometry (Figure 4C). Collectively these cross-linking data support the positioning of the rhodopsin C-tail in the crystal structure.Figure 4Validation of the Interface between Rhodopsin C-Tail and Arrestin by Disulfide Cross-LinkingShow full captionCrystal structures showing interface residues (left), and western blots showing disulfide cross-linking data (right). Black asterisk, arrestin; arrowhead, rhodopsin-arrestin cross-linking product.(A) The disulfide cross-linking between D330 of rhodopsin and K167 of arrestin, D331 of rhodopsin and R19 of arrestin, and E332 of rhodopsin and R19, K167, and K168 of arrestin.(B) The disulfide cross-linking between A333, S334, and A335 of rhodopsin and R19 of arrestin.(C) The disulfide cross-linking between T336 of rhodopsin and K16 and R19 of arrestin, and E341 of rhodopsin and K111 of arrestin.See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Crystal structures showing interface residues (left), and western blots showing disulfide cross-linking data (right). Black asterisk, arrestin; arrowhead, rhodopsin-arrestin cross-linking product. (A) The disulfide cross-linking between D330 of rhodopsin and K167 of arrestin, D331 of rhodopsin and R19 of arrestin, and E332 of rhodopsin and R19, K167, and K168 of arrestin. (B) The disulfide cross-linking between A333, S334, and A335 of rhodopsin and R19 of arrestin. (C) The disulfide cross-linking between T336 of rhodopsin and K16 and R19 of arrestin, and E341 of rhodopsin and K111 of arrestin. See also Figure S3. The interface revealed by the crystal structure was also confirmed by hydrogen-deuterium exchange mass spectrometry (HDX), which probes the dynamics of protein-protein interaction in solution (West et al., 2013West G.M. Pascal B.D. Ng L.M. Soon F.F. Melcher K. Xu H.E. Chalmers M.J. Griffin P.R. Protein conformation ensembles monitored by HDX reveal a structural rationale for abscisic acid signaling protein affinities and activities.Structure. 2013; 21: 229-235Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Compared to the free pre-activated arrestin, the binding of rhodopsin protects arrestin against hydrogen-deuterium exchange in several regions (Figure S3B). Notably, the arrestin regions protected from hydrogen-deuterium exchange include the N-terminal β strands (residues 11–52 and 115–120) and helix I (residues 104–110) (Figure S3B). The protection of these structural elements in the arrestin N-domain could not be explained by our previous structure that lacks the rhodopsin C-tail (Kang et al., 2015Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. et al.Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (583) Google Scholar), but it is consistent with the structure we report here, in which these arrestin regions are stabilized by binding to the C-terminal tail of rhodopsin (Figure S3C). We further analyzed the conformational stability within the β sheet formed by the rhodopsin C-terminal tail and the arrestin N terminus by performing two independent, three microsecond-long all-atom molecular dynamics simulations. Throughout both simulations, the charged interaction network connecting the phosphorylated T336 and S338 and the negatively charged E341 of rhodopsin with the positively charged arrestin residues comprising pockets A, B, and C was well maintained with average interaction frequencies upward of 80% for most crystallographic contacts (Figures 5A, 5B, and S4A). Overall, the RMSDs of pT336, pS338, and E341 generally remained within 3.0 Å of their crystallographic positions, consistent with maintenance of the interwoven ionic network, while residues D330 and S334 located N-terminally to pT336 exhibited a number of metastable states whose RMSDs reached values upward of 19 Å (Figure 5C). This differential stability of the C-tail was further characterized by the root-mean-square fluctuation (RMSF) of the C-tail alpha carbons around their average simulation positions. By defining an RMSF cut-off of 1.7 Å, both simulations suggest a key interaction range comprising C-tail residues A335–E341 (Figures 5D and 5E). Subsequent comparison of average simulation B-factors calculated from RMSF to the XFEL room temperature structure B-factors exhibits a remarkable agreement of stability within the key interactions mediated by the C-tail residues A335–E341 (Figure S4B).Figure S4Interaction Network and Range between the Rhodopsin C-Tail and Arrestin, Related to Figure 5Show full caption(A) Detailed analysis of the bonding network between pT336, pS338 and E341 and key residues comprising arrestin’s three phosphate binding pockets for both simulations. Only interactions with frequencies greater than 5% are shown. Residues marked with ∗ indicated backbone interactions.(B) Simulation B-factors (left) derived from Cα RMSFs agree with room temperature XFEL structure B-factors (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Detailed analysis of the bonding network between pT336, pS338 and E341 and key residues comprising arrestin’s three phosphate binding pockets for both simulations. Only interactions with frequencies greater than 5% are shown. Residues marked with ∗ indicated backbone interactions. (B) Simulation B-factors (left) derived from Cα RMSFs agree with room temperature XFEL structure B-factors (right). Phosphorylation-dependent recruitment of arrestins is a common feature for GPCRs (Lefkowitz and Shenoy, 2005Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by beta-arrestins.Science. 2005; 308: 512-517Crossref PubMed Scopus (1427) Google Scholar, Shukla et al., 2011Shukla A.K. Xiao K. Lefkowitz R.J. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling.Trends Biochem. Sci. 2011; 36: 457-469Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). The structure of the phosphorylated rhodopsin-arrestin complex, together with the previous structure of β-arrestin-1 in complex with phosphorylated V2R C-tail peptide (Shukla et al., 2013Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. et al.Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.Nature. 2013; 497: 137-141Crossref PubMed Scopus (321) Google Scholar), provides mechanistic insights into phosphorylation-dependent arrestin recruitment by GPCRs. Superposition of the V2R-β-arrestin-1 structure (PDB: 4JQI) with our rhodopsin-arrestin complex structure reveals a noticeable similarity between the β strand of V2R (residues 360–365) and the C-terminal β strand (residues 338–343) of rhodopsin (Figures 6A and 6B). The phosphorylated residues S357 and T360 of the V2R C-tail are nearly superposable with the phosphorylated T336 and S338 of rhodopsin C-tail (Figure 6B). In addition, the ionic interaction between the phosphorylated S363 of V2R and K107 of β-arrestin-1 is comparable to the interaction of rhodopsin E341 with K111 of visual arrestin (Figures 6B and S5A). The V2R residues N-terminal to the phosphorylated S357, however, are located differently compared to those preceeding T336 in rhodopsin. The V2R residues N-terminal to phosphorylated S357 are positioned close to a location corresponding to the middle loop and the finger loop of β-arrestin-1 (Figures 6B and S5A). As revealed by electron microscopic studies of β2AR-V2R C-tail chimeric receptor in complex with β-arrestin-1, arrestin can bind either to the central cytoplasmic cavity of the receptor TM bundle in a fully engaged state or to the receptor’s C-terminal tail in a partially tail-engaged state (Shukla et al., 2014Shukla A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. et al.Visualization of arrestin recruitment by a G-protein-coupled receptor.Nature. 2014; 512: 218-222Crossref PubMed Scopus (365) Google Scholar). The position of those V2R residues would clash with the corresponding residues in the finger loop of visual arrestin in the fully engaged state, thus the structure of β-arrestin-1 in complex with the phosphorylated V2R C-tail likely represents a tail-engaged state, whereas the structure of the phosphorylated rhodopsin-arrestin complex represents the fully engaged state.Figure S5Receptor C-Tail-Arrestin Interfaces Are Conserved, Related to Figure 6Show full caption(A) Binding of V2R C-tail (magenta) to the β-arrestin N-terminal surface (PDB code: 4JQI). Blue indicates positive charges, and red negative charges at the arrestin surface. The phospho-residues binding to the" @default.
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• W2739933313 modified "2023-10-16" @default.
• W2739933313 title "Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors" @default.
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• W2739933313 doi "https://doi.org/10.1016/j.cell.2017.07.002" @default.
• W2739933313 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5567868" @default.
• W2739933313 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28753425" @default.
• W2739933313 hasPublicationYear "2017" @default.
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