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Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol

Abstract

Recent high-pressure NMR results indicate that the preactive conformation of the β1-adrenergic receptor (β1AR) harbours completely empty cavities of ~100 Å3 volume, which disappear in the active conformation of the receptor. Here we have localized these cavities using X-ray crystallography of xenon-derivatized β1AR crystals. One of the cavities is in direct contact with the cholesterol-binding pocket. Solution NMR shows that addition of the cholesterol analogue cholesteryl hemisuccinate impedes the formation of the active conformation of detergent-solubilized β1AR by blocking conserved G protein-coupled receptor microswitches, concomitant with an affinity reduction of both isoprenaline and G protein-mimicking nanobody Nb80 for β1AR detected by isothermal titration calorimetry. This wedge-like action explains the function of cholesterol as a negative allosteric modulator of β1AR. A detailed understanding of G protein-coupled receptor regulation by cholesterol by filling of a dry void and the easy scouting for such voids by xenon may provide new routes for the development of allosteric drugs.

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Fig. 1: Xenon derivatization of TS-β1AR.
Fig. 2: High-pressure NMR analysis of agonist-bound β1AR labelled with 15N-valine in the presence or absence of CHS.
Fig. 3: CHS shifts β1AR from the active to the preactive conformation.
Fig. 4: Effect of CHS on the thermodynamics of isoprenaline and Nb80 binding to YY-β1AR-V129I.
Fig. 5: Structural basis of the allosteric inhibition of β1AR by CHS.

Data availability

NMR spectra and ITC raw data, as well as structure factors, phases and density maps derived from the anomalous scattering data of the four xenon-derivatized isoprenaline·β1AR crystals and the β1AR structure derived from the first crystal have been deposited in the Zenodo repository under https://doi.org/10.5281/zenodo.4926013. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Swiss National Science Foundation (Grants CRSK-3_195592 to L.A.A. and 31-149927, 31-173089 and 31-201270 to S.G.). We gratefully acknowledge M. Schaffhauser, P. Schlenker and R. Strittmatter (Biozentrum Central Mechanical Workshop) as well as S. Saner (Biozentrum Central Electronics Workshop) for designing and building the xenon pressure chamber apparatus, the Paul Scherrer Institut, Villigen, Switzerland for synchrotron radiation beamtime at beamline PXIII, the Biozentrum Biophysics Facility for access to their instruments, J. Steyaert for the generous gift of the Nb80 plasmid, as well as H.-J. Sass, C. Tate, T. Maier and T. Schirmer for helpful discussions.

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L.A.A. and S.G. conceived the study. L.A.A. and A.G. expressed and purified proteins and recorded NMR spectra. L.A.A. recorded high-pressure NMR experiments. L.A.A. analysed and interpreted the NMR data. R.D.T. and S.E. recorded the X-ray data. R.D.T. analysed the X-ray data. T.M. and T.S. performed and analysed ITC experiments. L.A.A., R.D.T. and S.G. wrote the manuscript.

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Correspondence to Layara Akemi Abiko or Stephan Grzesiek.

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Supplementary Table 1

Numerical source of 1H NMR resonance intensities plotted in Supplementary Fig. 3b.

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Numerical Source Data for Fig. 3c.

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Abiko, L.A., Dias Teixeira, R., Engilberge, S. et al. Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol. Nat. Chem. 14, 1133–1141 (2022). https://doi.org/10.1038/s41557-022-01009-9

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