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Crystal structure of a common GPCR-binding interface for G protein and arrestin.

Szczepek M, Beyrière F, Hofmann KP, Elgeti M, Kazmin R, Rose A, Bartl FJ, von Stetten D, Heck M, Sommer ME, Hildebrand PW, Scheerer P - Nat Commun (2014)

Bottom Line: Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin.For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice.Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.

ABSTRACT
G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

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Overall structure of the Ops*–ArrFL-1 complex.(a) The active receptor Ops* (orange) and the ArrFL-1 peptide (purple) are shown in ribbon representation, and the ArrFL-1 side chains are shown as sticks. Oligosaccharides at Asn15NT (pink) and a palmitoyl chain at Cys3238.60 (black) are presented as sticks. The detergent molecule, β-D-octylglucopyranoside (blue), is shown within the ligand-binding pocket as sticks. (b) Side view and (c) top view of the binding crevice of Ops*. Major hydrogen bonding interactions of Ops* with ArrFL-1 are shown between Leu77 and Lys3118.48 from the NPxxY(x)5,6F motif (green), and Met75 and Arg1353.50 from the E(D)RY motif (blue). (d) Additional rotated top view of the binding crevice of Ops*. Hydrophobic interactions between ArrFL-1 and residues in the binding interface of Ops* protein moiety are shown as sticks and spheres (light-blue). Arg1353.50 of TM3, Val1383.53, Lys141CL2 of CL2, Glu2496.32, Val2506.33 of TM6 and Asn3108.47, Lys3118.48 of TM7/H8 are in van der Waals contact to ArrFL-1 (that is, separated by less than 4 Å).
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f2: Overall structure of the Ops*–ArrFL-1 complex.(a) The active receptor Ops* (orange) and the ArrFL-1 peptide (purple) are shown in ribbon representation, and the ArrFL-1 side chains are shown as sticks. Oligosaccharides at Asn15NT (pink) and a palmitoyl chain at Cys3238.60 (black) are presented as sticks. The detergent molecule, β-D-octylglucopyranoside (blue), is shown within the ligand-binding pocket as sticks. (b) Side view and (c) top view of the binding crevice of Ops*. Major hydrogen bonding interactions of Ops* with ArrFL-1 are shown between Leu77 and Lys3118.48 from the NPxxY(x)5,6F motif (green), and Met75 and Arg1353.50 from the E(D)RY motif (blue). (d) Additional rotated top view of the binding crevice of Ops*. Hydrophobic interactions between ArrFL-1 and residues in the binding interface of Ops* protein moiety are shown as sticks and spheres (light-blue). Arg1353.50 of TM3, Val1383.53, Lys141CL2 of CL2, Glu2496.32, Val2506.33 of TM6 and Asn3108.47, Lys3118.48 of TM7/H8 are in van der Waals contact to ArrFL-1 (that is, separated by less than 4 Å).

Mentions: We co-crystallized R* (light-activated rhodopsin (Meta II) or the active form of the apoprotein, Ops*) with ArrFL peptides derived from rod photoreceptor arrestin (67YGQEDIDVMGL77=ArrFL-1), β-arrestin-1 and β-arrestin-2 (63/64YGREDLDVLGL73/74=ArrFL-2/3). Our first strategy was to illuminate a mixture of rhodopsin and ArrFL peptides directly before crystallization (Supplementary Figs 2 and 3). The resulting crystal structures (Meta II–ArrFL) showed weak electron density for both the all-trans-retinal ligand and the peptides (only three residues from the C-terminus were resolved). This problem was most likely due to structural heterogeneity within the crystal, which we overcame by co-crystallizating Ops* and ArrFL peptides. Best results were obtained with ArrFL-1, which diffracted up to 2.75 Å resolution (Fig. 2, Table 1, Supplementary Figs 2–4). The Ops*–ArrFL-1 structure contains amino acids 2–326 of opsin, including the 7-TM helices connected by extracellular (EL1–EL3) and cytoplasmic (CL1–CL3) loops, and the cytoplasmic helix 8 (H8), which runs along the membrane surface (Fig. 2a, Supplementary Fig. 4). The last 22 C-terminal amino acids of Ops* were not resolved, presumably due to their high flexibility. The atomic structure of the ArrFL-1 peptide (residues 71–77) was solved from a continuous electron density located within the cytoplasmic crevice of R* (Supplementary Fig. 4), whereas the N-terminal extended part of the peptide (residues 71–73) showed weaker electron density (Supplementary Table 1). Well-defined electron density for a detergent molecule, β-D-octylglucopyranoside, is present within the ligand-binding pocket of R* (Supplementary Fig. 5)20. Note that the overall structure of the receptor in all Meta II–ArrFL peptide complexes are essentially the same as the Ops*–ArrFL-1 structure.


Crystal structure of a common GPCR-binding interface for G protein and arrestin.

Szczepek M, Beyrière F, Hofmann KP, Elgeti M, Kazmin R, Rose A, Bartl FJ, von Stetten D, Heck M, Sommer ME, Hildebrand PW, Scheerer P - Nat Commun (2014)

Overall structure of the Ops*–ArrFL-1 complex.(a) The active receptor Ops* (orange) and the ArrFL-1 peptide (purple) are shown in ribbon representation, and the ArrFL-1 side chains are shown as sticks. Oligosaccharides at Asn15NT (pink) and a palmitoyl chain at Cys3238.60 (black) are presented as sticks. The detergent molecule, β-D-octylglucopyranoside (blue), is shown within the ligand-binding pocket as sticks. (b) Side view and (c) top view of the binding crevice of Ops*. Major hydrogen bonding interactions of Ops* with ArrFL-1 are shown between Leu77 and Lys3118.48 from the NPxxY(x)5,6F motif (green), and Met75 and Arg1353.50 from the E(D)RY motif (blue). (d) Additional rotated top view of the binding crevice of Ops*. Hydrophobic interactions between ArrFL-1 and residues in the binding interface of Ops* protein moiety are shown as sticks and spheres (light-blue). Arg1353.50 of TM3, Val1383.53, Lys141CL2 of CL2, Glu2496.32, Val2506.33 of TM6 and Asn3108.47, Lys3118.48 of TM7/H8 are in van der Waals contact to ArrFL-1 (that is, separated by less than 4 Å).
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f2: Overall structure of the Ops*–ArrFL-1 complex.(a) The active receptor Ops* (orange) and the ArrFL-1 peptide (purple) are shown in ribbon representation, and the ArrFL-1 side chains are shown as sticks. Oligosaccharides at Asn15NT (pink) and a palmitoyl chain at Cys3238.60 (black) are presented as sticks. The detergent molecule, β-D-octylglucopyranoside (blue), is shown within the ligand-binding pocket as sticks. (b) Side view and (c) top view of the binding crevice of Ops*. Major hydrogen bonding interactions of Ops* with ArrFL-1 are shown between Leu77 and Lys3118.48 from the NPxxY(x)5,6F motif (green), and Met75 and Arg1353.50 from the E(D)RY motif (blue). (d) Additional rotated top view of the binding crevice of Ops*. Hydrophobic interactions between ArrFL-1 and residues in the binding interface of Ops* protein moiety are shown as sticks and spheres (light-blue). Arg1353.50 of TM3, Val1383.53, Lys141CL2 of CL2, Glu2496.32, Val2506.33 of TM6 and Asn3108.47, Lys3118.48 of TM7/H8 are in van der Waals contact to ArrFL-1 (that is, separated by less than 4 Å).
Mentions: We co-crystallized R* (light-activated rhodopsin (Meta II) or the active form of the apoprotein, Ops*) with ArrFL peptides derived from rod photoreceptor arrestin (67YGQEDIDVMGL77=ArrFL-1), β-arrestin-1 and β-arrestin-2 (63/64YGREDLDVLGL73/74=ArrFL-2/3). Our first strategy was to illuminate a mixture of rhodopsin and ArrFL peptides directly before crystallization (Supplementary Figs 2 and 3). The resulting crystal structures (Meta II–ArrFL) showed weak electron density for both the all-trans-retinal ligand and the peptides (only three residues from the C-terminus were resolved). This problem was most likely due to structural heterogeneity within the crystal, which we overcame by co-crystallizating Ops* and ArrFL peptides. Best results were obtained with ArrFL-1, which diffracted up to 2.75 Å resolution (Fig. 2, Table 1, Supplementary Figs 2–4). The Ops*–ArrFL-1 structure contains amino acids 2–326 of opsin, including the 7-TM helices connected by extracellular (EL1–EL3) and cytoplasmic (CL1–CL3) loops, and the cytoplasmic helix 8 (H8), which runs along the membrane surface (Fig. 2a, Supplementary Fig. 4). The last 22 C-terminal amino acids of Ops* were not resolved, presumably due to their high flexibility. The atomic structure of the ArrFL-1 peptide (residues 71–77) was solved from a continuous electron density located within the cytoplasmic crevice of R* (Supplementary Fig. 4), whereas the N-terminal extended part of the peptide (residues 71–73) showed weaker electron density (Supplementary Table 1). Well-defined electron density for a detergent molecule, β-D-octylglucopyranoside, is present within the ligand-binding pocket of R* (Supplementary Fig. 5)20. Note that the overall structure of the receptor in all Meta II–ArrFL peptide complexes are essentially the same as the Ops*–ArrFL-1 structure.

Bottom Line: Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin.For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice.Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.

ABSTRACT
G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

Show MeSH