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Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs


The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known1,2. Here we determine binding sites, bound conformations and specific drug–receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation–π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, ‘orthosteric’ ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator’s allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

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Figure 1: Structurally diverse allosteric modulators bind spontaneously to the human M2 muscarinic acetylcholine receptor in simulation, revealing a common binding mode.
Figure 2: Experimental validation of computationally derived binding modes.
Figure 3: Mechanisms of positive and negative allosteric modulation.


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We thank T. Mildorf, A. Kruse and B. Kobilka for comments; J. Klepeis, B. Gregersen, J.-L. Li, K. Palmo, A. Donchev and particularly A. Taube for advice and support related to force fields and quantum mechanical calculations; Z. Fan for assistance with statistical analysis; A. Lerer and T. O’Donnell for assistance with simulation and analysis software; A. Philippsen for creating the video; A. Stewart for assistance with mutagenesis and cell line generation; K. Ban and J. Harjani for assistance with chemical synthesis; J. Swarbrick for recording and analysing the two-dimensional NMR data; B. Sleebs and S. Marcuccio for provision of synthetic reagents; J. Dang for advice on analytical chemistry; and M. Kirk and R. Kastleman for editorial assistance. Portions of this work were financed by Program Grant no. 519461 from the National Health and Medical Research Council (NHMRC) of Australia, with synthetic chemistry infrastructure support from the Australian Federal Education Investment Fund Super Science Initiative and Victoria’s Science Agenda Investment Fund. A.C. and P.M.S. are Principal Research Fellows of the NHMRC; J.B.B. is a Senior Research Fellow of the NHMRC; J.R.L. is a Career Development Awardee of the NHMRC.

Author information




R.O.D. conceived this study and, with D.E.S., oversaw molecular dynamics simulations and analysis. R.O.D., H.F.G., D.W.B., J.R.V., A.C.P. and D.H.A. designed and analysed molecular dynamics simulations. H.F.G., J.R.V., A.C.P. and D.H.A. performed molecular dynamics simulations. R.O.D., H.F.G., D.W.B. and J.R.V. performed computational design of receptor mutants and of the modulator 4P-C7/3-phth. C.V. performed all biological assays and, with J.R.V. and A.C., analysed experimental data. M.C. and J.R.L. performed mutagenesis and generated the stable cell lines. J.B.B. and R.R. designed, planned and executed the synthesis of 4P-C7/3-phth, with active input from D.W.B. P.M.S. and A.C. supervised the cell-based biological studies. R.O.D., H.F.G., D.W.B., A.C. and D.E.S. wrote the manuscript. R.O.D., A.C. and D.E.S. supervised the overall research.

Corresponding authors

Correspondence to Ron O. Dror, Arthur Christopoulos or David E. Shaw.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-25, Supplementary Tables 1-8, Supplementary Methods and additional references. (PDF 5767 kb)

The allosteric modulator C7/3-phth binds spontaneously to the M2 receptor in an unbiased molecular dynamics simulation

For clarity, the lipid bilayer, ions, and water molecules are not shown. The video speeds up 44-fold after the modulator binds at 120 ns; the amount of simulated time between successive video frames is 1.08 ns before this point and 47.5 ns afterwards. The Cartesian components of the protein Cα positions were smoothed using Fourier-based Gaussian smoothing (σ = 7.2 ns). Ligand coordinates were not smoothed before the ligand bound, but once it bound, both the Cartesian components of its atom positions and its internal angles were smoothed using Gaussian filters (σ = 7.2 ns). The video was created using OpenStructure17. This is simulation 1 under condition A (Supplementary Table 2). (MP4 2760 kb)

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Dror, R., Green, H., Valant, C. et al. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature 503, 295–299 (2013).

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