.2020 Feb 21;367(6480):881-887.

doi: 10.1126/science.aaz0326.

Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor

Carl-Mikael Suomivuori^#^{1 2 3 4}, Naomi R Latorraca^#^{1 2 3 4 5}, Laura M Wingler^{6 7}, Stephan Eismann^{1 2 3 4 8}, Matthew C King^{1 2 3 4}, Alissa L W Kleinhenz^{6 7 9}, Meredith A Skiba¹⁰, Dean P Staus^{6 7}, Andrew C Kruse¹⁰, Robert J Lefkowitz^{6 7 11}, Ron O Dror^{12 2 3 4 5}

Affiliations

¹ Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
² Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁴ Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA 94305, USA.
⁵ Biophysics Program, Stanford University, Stanford, CA 94305, USA.
⁶ Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
⁷ Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
⁸ Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.
⁹ School of Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
¹⁰ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
¹² Department of Computer Science, Stanford University, Stanford, CA 94305, USA. ron.dror@stanford.edu.

^# Contributed equally.

PMID:32079767
PMCID: PMC7259329
DOI: 10.1126/science.aaz0326

Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor

Carl-Mikael Suomivuori et al. Science.2020.

.2020 Feb 21;367(6480):881-887.

doi: 10.1126/science.aaz0326.

Authors

Affiliations

¹ Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
² Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁴ Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA 94305, USA.
⁵ Biophysics Program, Stanford University, Stanford, CA 94305, USA.
⁶ Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
⁷ Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
⁸ Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.
⁹ School of Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
¹⁰ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
¹² Department of Computer Science, Stanford University, Stanford, CA 94305, USA. ron.dror@stanford.edu.

^# Contributed equally.

PMID:32079767
PMCID: PMC7259329
DOI: 10.1126/science.aaz0326

Abstract

Biased signaling, in which different ligands that bind to the same G protein-coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. The receptor adopts two major signaling conformations, one of which couples almost exclusively to arrestin, whereas the other also couples effectively to a G protein. A long-range allosteric network allows ligands in the extracellular binding pocket to favor either of the two intracellular conformations. Guided by this computationally determined mechanism, we designed ligands with desired signaling profiles.

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Conflict of interest statement

Competing interests: R.J.L. is a founder and stockholder of Trevena and a director of Lexicon Pharmaceuticals. A.C.K. is an advisor for the Institute for Protein Innovation, a non-profit research institute.

Figures

**Fig. 1.. In simulation, AT₁R adopts two major signaling conformations.**
In a representative simulation with the nanobody removed,AT₁R first adopts the “alternative” conformation andthen transitions to the “canonical active” conformation. Duringthis transition, TM7 twists above its proline kink, leading the intracellularportion of TM7 to shift away from TMs 2 and 3. The twisting motion causes N1.50to switch its preferred hydrogen bond acceptor from C7.47 to N7.46 (top trace,distance between N1.50 side-chain nitrogen and N7.46 backbone oxygen). Theconformational transition also leads to rearrangements of side chains, includingthose of Y7.53 and R3.50, which are oriented downward (toward the intracellularside) in the alternative conformation and more upward in the canonical activeconformation (bottom trace; see also Fig. S3). Thick traces representmoving averages, while thin traces represent original, unsmoothed values (seeMethods). Dashedhorizontal lines indicate values for the nanobody-bound crystal structures.

**Fig. 2.. Structural models suggest that the alternative conformation couples preferentially to β-arrestins, while the canonical active conformation couples well to both Gq and β-arrestins.**
In the alternative conformation, R3.50 adopts an orientation thatclashes with a tyrosine on the α5 helix of Gq (top right). The bottom rowshows models with β-arrestin 1; models with β-arrestin 2 yieldessentially identical results (see Methods).

**Fig. 3. Allosteric network allows ligands to favor either intracellular conformation.**
**(A)** Three coupled regions of AT₁R connect theligand-binding pocket to the intracellular surface. Key residues for the threeregions are shown in turquoise, orange, and purple, respectively.(B) Mechanism for coupling between the three regions. Insimulations that transition from the alternative conformation to the canonicalactive conformation, a rotation of TM3 at the binding pocket triggers subsequentrearrangements in other regions of the protein (top row, left and middlepanels). Such transitions typically begin when L3.36 moves closer to TM2 (topturquoise trace) and N3.35 flips outward from the helical bundle (bottomturquoise trace). The outward displacement of N3.35 creates space for F2.53 toswitch positions with Y7.43 (orange traces) (top row, middle and right panels).The motion of Y7.43 leads TM7 to twist just above its proline kink (bottom row,all panels), such that N7.46 replaces C7.47 as the hydrogen bonding partner ofN1.50 (purple traces). This TM7 twist also leads to an outward shift of TM7 onthe intracellular side, as measured by increasing P7.50–L2.46Cα–Cα distances; these distances are shown in blackrectangles in the bottom row of molecular renderings. See also Fig. S5.(C) Insimulation, the fraction of time spent in the alternative TM7 conformationcorrelates with the ligand’s bias profile. β-arrestin-biasedligands favor this conformation much more than Gq-biased ligands(P = 0.0006, two-sidedt-test; see Methods), and also morethan the balanced ligand AngII (P = 0.007), which favors itmore than Gq-biased ligands (P = 0.04). Reported values arebased on REMD simulations of AT₁R bound to each ligand. We performedtwo REMD simulations for AngII (with very similar results of 0.32 and 0.33,respectively), and one for each other ligand. Each REMD simulation consists of36 coupled MD simulations, each 3.6 μs in length (see Methods).

**Fig. 4.. Arrestin-biased, balanced, and G-protein-biased ligands favor distinct binding-pocket conformations.**
**(A)** Structures of representative β-arrestin-biased,balanced, and Gq-biased AT₁R ligands. All ligands studied in thiswork are shown in Fig.S1.(B) In comparison to β-arrestin-biasedligands, AngII drives L3.36 of the binding pocket toward its TM2-proximalposition, which favors the canonical active conformation, and Gq-biased ligandsdo so even more (P = 0.002 for β-arrestin-biased vs.Gq-biased ligands; see Methods) (left; the bar plot shows means and standard errors across5 independent 2-μs simulations per ligand). When the F8 residue of AngIIand Gq-biased ligands adopts a horizontal orientation, it pushes L3.36 towardthe TM2-proximal position (right; gray and pink dashed lines indicate values forTRV023- and AngII-bound structures, respectively; traces are for a simulationwith AngII bound, and additional simulation traces are shown in Fig. S6). RMSD is root-mean-squaredeviation (see Methods).(C) AngII and β-arrestin-biased ligandsstabilize more inward TM6 positions in the binding pocket compared to positionsfavored by Gq-biased ligands (P = 0.005 for Gq-biased ligandsvs. AngII;P = 0.001 for Gq-biased ligands vs.β-arrestin-biased ligands), as shown by the box plot at left (boxesextend from 25^th to 75^th percentile of simulation frames),because the ligand R2 residue interacts with D6.58 and D7.32 (right; dashedlines correspond to distance plotted at left). The more outward position of theextracellular portion of TM6 observed for Gq-biased ligands allows their F8residue to adopt a horizontal orientation more frequently than that of AngII(see also Fig. S7).

**Fig. 5.. Rational design of ligands with desired signaling profiles.**
**(A)** The addition of a single connecting methylene group tothe phenyl moiety of AngII ligand restrains the C-terminal ring to remainvertical, producing a strongly arrestin-biased ligand, Ind8-AngII, which barelycouples to Gq.(B) S1I8 has partial activity toward both theGq-mediated and β-arrestin-mediated pathways. S1A2I8, which was designedto increase Gq signaling relative to S1I8 by favoring outward motion of TM6 inthe binding pocket, shows increased Gq efficacy without any correspondingincrease in β-arrestin efficacy. S1A2I8 is the R2A mutant of S1I8. Errorbars represent standard error from 3–4 independent experiments. See alsoTable S2.

**Fig. 6.. Observed intracellular conformations of AT₁R.**
Simulations indicate that AT₁R adopts at least five distinctconformations that differ substantially in the intracellular positions of TM6and TM7 and may have distinct cellular signaling profiles. In the inactiveconformation (gray; illustrated by crystal structure (24)), TM6 occludes the transducer-binding pocket,hindering coupling to either G proteins or arrestins. The remaining fourconformations all exhibit more outward positions of TM6 but differ substantiallyin the conformation of TM7, as illustrated by representative frames from ourAT₁R simulations. In the middle row, we show TMs 6 and 7 of thesefour conformations (colors) overlaid on all TMs of the inactive structure(gray). Our results suggest that in the alternative conformation (orange), theintracellular surface hinders coupling to G proteins but allows for arrestincoupling, whereas the canonical active conformation (blue) couples to both Gproteins and arrestins. Two other conformations that AT₁R adopts insimulation—the TM6-bent and non-canonical conformations, in purple andpink, respectively—have been observed in G-protein-bound structures ofother GPCRs but might hinder arrestin coupling by increasing the volume of thetransducer-binding pocket, preventing the arrestin finger loop from packingtightly against this pocket.

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References

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Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor

Affiliations

Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

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Substances

Grants and funding

LinkOut - more resources

Full Text Sources

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Molecular Biology Databases