Limits...
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.

Show MeSH
Structural and functional comparison of GtαCT and ArrFL-1.(a) Side view of the active receptor Ops* crevice (black ribbons and sticks) with bound GtαCT-HA peptide (blue ribbon and sticks; PDB entry 3DQB). (b) Side view of the active receptor Ops* crevice (orange ribbons and sticks) with bound ArrFL-1 peptide (purple ribbon and sticks). (c) Stabilization of Meta II by the high-affinity peptide GtαCT-HA and (d) ArrFL-1 peptide. (e) Competition against rod photoreceptor arrestin for binding to phosphorylated opsin by GtαCT-HA and (f) ArrFL-1 peptide. In panels c–f, data points from independent experiments are represented as differently shaped symbols. The peptide titration experiments which measure Meta II-stabilization (c,d and Supplementary Fig. 9a,b,c) yield an apparent KD, from which the true KD value of peptide binding to Meta II can be derived. Further experimental details are given in the Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199108&req=5

f4: Structural and functional comparison of GtαCT and ArrFL-1.(a) Side view of the active receptor Ops* crevice (black ribbons and sticks) with bound GtαCT-HA peptide (blue ribbon and sticks; PDB entry 3DQB). (b) Side view of the active receptor Ops* crevice (orange ribbons and sticks) with bound ArrFL-1 peptide (purple ribbon and sticks). (c) Stabilization of Meta II by the high-affinity peptide GtαCT-HA and (d) ArrFL-1 peptide. (e) Competition against rod photoreceptor arrestin for binding to phosphorylated opsin by GtαCT-HA and (f) ArrFL-1 peptide. In panels c–f, data points from independent experiments are represented as differently shaped symbols. The peptide titration experiments which measure Meta II-stabilization (c,d and Supplementary Fig. 9a,b,c) yield an apparent KD, from which the true KD value of peptide binding to Meta II can be derived. Further experimental details are given in the Methods.

Mentions: To verify the functional relevance of the observed peptide binding to the receptor crevice (Fig. 4a, b), we applied three different well-established functional assays. These assays were performed in native disc membranes and not the detergent system used for crystallization, as the native membrane offers many advantages to observe the interactions of peptides with the different functional forms of rhodopsin. In the first assay, we light-activated rhodopsin under conditions favouring Meta I, the inactive precursor of the active species Meta II. Meta I and Meta II can be differentiated by ultraviolet-visible absorption spectroscopy due to their different absorbance maxima. Like both full-length rod photoreceptor arrestin (ref. 30) and GtαCT31, ArrFL-1 stabilized the 380-nm-absorbing Meta II species over Meta I, showing that ArrFL-1 functionally binds the active state of the receptor. Titration analysis indicated KD values of 2.4 μM and 0.8 mM for GtαCT-HA (high-affinity version) and ArrFL-1, respectively (Fig. 4c, d). For comparison, the KD values of the wild-type GtαCT and ArrFL-2/3 are 0.18 and 1.3 mM, respectively (Supplementary Fig. 9). In the second assay, we measured the ability of GtαCT and ArrFL peptides to compete against full-length rod photoreceptor arrestin for binding to phosphorylated opsin (OpsP) in membranes (Fig. 4e,f, Supplementary Fig. 9). The competition data yield KD values of 320 μM, 880 μM, 1.7 mM and 2.4 mM for peptides GtαCT-HA, wild-type GtαCT, ArrFL-1 and ArrFL-2/3, respectively. Importantly, these data indicate that full-length rod photoreceptor arrestin and ArrFL have a common receptor-binding site. The peptide derived from GsαCT from the Gs family, which does not share the common sequence motif with ArrFL and GtαCT, did not stabilize Meta II and showed no competition for arrestin binding to phosphorylated opsin (Supplementary Fig. 9). In the third assay, Fourier transform infrared (FTIR) spectroscopy was used to monitor the effects of Meta II–ArrFL-1 complex formation in native membranes resulting in characteristic bands, which mostly appear in the structurally sensitive amide I and amide II regions of the FTIR spectrum3233 (Supplementary Fig. 10). The effect of ArrFL-1 and ArrFL-2/3 on the R* characteristic difference spectrum is similar to GtαCT, although the data suggest a somewhat smaller influence on the peptide structure due to binding32. The GsαCT shows essentially no effect on the difference spectrum, in accordance with its lack of binding in the other assays. In further FTIR experiments, we investigated the R135L mutant and its capability of binding ArrFL-1 peptide (Supplementary Fig. 10). We observed that the characteristic arginine bands are missing upon binding, which suggest an involvement of Arg135 in ArrFL-1 binding under these conditions. Altogether, FTIR data confirm that peptides with the consensus sequence (E/D)x(I/L)xxxGL specifically bind and stabilize the active conformation of the receptor in a very similar manner as GtαCT. It remains to be determined whether, like GtαCT, ArrFL-1 discriminates between different conformational substates of Meta II (ref. 33).


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)

Structural and functional comparison of GtαCT and ArrFL-1.(a) Side view of the active receptor Ops* crevice (black ribbons and sticks) with bound GtαCT-HA peptide (blue ribbon and sticks; PDB entry 3DQB). (b) Side view of the active receptor Ops* crevice (orange ribbons and sticks) with bound ArrFL-1 peptide (purple ribbon and sticks). (c) Stabilization of Meta II by the high-affinity peptide GtαCT-HA and (d) ArrFL-1 peptide. (e) Competition against rod photoreceptor arrestin for binding to phosphorylated opsin by GtαCT-HA and (f) ArrFL-1 peptide. In panels c–f, data points from independent experiments are represented as differently shaped symbols. The peptide titration experiments which measure Meta II-stabilization (c,d and Supplementary Fig. 9a,b,c) yield an apparent KD, from which the true KD value of peptide binding to Meta II can be derived. Further experimental details are given in the Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199108&req=5

f4: Structural and functional comparison of GtαCT and ArrFL-1.(a) Side view of the active receptor Ops* crevice (black ribbons and sticks) with bound GtαCT-HA peptide (blue ribbon and sticks; PDB entry 3DQB). (b) Side view of the active receptor Ops* crevice (orange ribbons and sticks) with bound ArrFL-1 peptide (purple ribbon and sticks). (c) Stabilization of Meta II by the high-affinity peptide GtαCT-HA and (d) ArrFL-1 peptide. (e) Competition against rod photoreceptor arrestin for binding to phosphorylated opsin by GtαCT-HA and (f) ArrFL-1 peptide. In panels c–f, data points from independent experiments are represented as differently shaped symbols. The peptide titration experiments which measure Meta II-stabilization (c,d and Supplementary Fig. 9a,b,c) yield an apparent KD, from which the true KD value of peptide binding to Meta II can be derived. Further experimental details are given in the Methods.
Mentions: To verify the functional relevance of the observed peptide binding to the receptor crevice (Fig. 4a, b), we applied three different well-established functional assays. These assays were performed in native disc membranes and not the detergent system used for crystallization, as the native membrane offers many advantages to observe the interactions of peptides with the different functional forms of rhodopsin. In the first assay, we light-activated rhodopsin under conditions favouring Meta I, the inactive precursor of the active species Meta II. Meta I and Meta II can be differentiated by ultraviolet-visible absorption spectroscopy due to their different absorbance maxima. Like both full-length rod photoreceptor arrestin (ref. 30) and GtαCT31, ArrFL-1 stabilized the 380-nm-absorbing Meta II species over Meta I, showing that ArrFL-1 functionally binds the active state of the receptor. Titration analysis indicated KD values of 2.4 μM and 0.8 mM for GtαCT-HA (high-affinity version) and ArrFL-1, respectively (Fig. 4c, d). For comparison, the KD values of the wild-type GtαCT and ArrFL-2/3 are 0.18 and 1.3 mM, respectively (Supplementary Fig. 9). In the second assay, we measured the ability of GtαCT and ArrFL peptides to compete against full-length rod photoreceptor arrestin for binding to phosphorylated opsin (OpsP) in membranes (Fig. 4e,f, Supplementary Fig. 9). The competition data yield KD values of 320 μM, 880 μM, 1.7 mM and 2.4 mM for peptides GtαCT-HA, wild-type GtαCT, ArrFL-1 and ArrFL-2/3, respectively. Importantly, these data indicate that full-length rod photoreceptor arrestin and ArrFL have a common receptor-binding site. The peptide derived from GsαCT from the Gs family, which does not share the common sequence motif with ArrFL and GtαCT, did not stabilize Meta II and showed no competition for arrestin binding to phosphorylated opsin (Supplementary Fig. 9). In the third assay, Fourier transform infrared (FTIR) spectroscopy was used to monitor the effects of Meta II–ArrFL-1 complex formation in native membranes resulting in characteristic bands, which mostly appear in the structurally sensitive amide I and amide II regions of the FTIR spectrum3233 (Supplementary Fig. 10). The effect of ArrFL-1 and ArrFL-2/3 on the R* characteristic difference spectrum is similar to GtαCT, although the data suggest a somewhat smaller influence on the peptide structure due to binding32. The GsαCT shows essentially no effect on the difference spectrum, in accordance with its lack of binding in the other assays. In further FTIR experiments, we investigated the R135L mutant and its capability of binding ArrFL-1 peptide (Supplementary Fig. 10). We observed that the characteristic arginine bands are missing upon binding, which suggest an involvement of Arg135 in ArrFL-1 binding under these conditions. Altogether, FTIR data confirm that peptides with the consensus sequence (E/D)x(I/L)xxxGL specifically bind and stabilize the active conformation of the receptor in a very similar manner as GtαCT. It remains to be determined whether, like GtαCT, ArrFL-1 discriminates between different conformational substates of Meta II (ref. 33).

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