Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations |
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Authors: | Tomohiro Kimura Krishna Vukoti Diane L. Lynch Dow P. Hurst Alan Grossfield Michael C. Pitman Patricia H. Reggio Alexei A. Yeliseev Klaus Gawrisch |
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Affiliation: | 1. Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, , Bethesda, Maryland, 20892;2. Department of Chemistry and Biochemistry, University of North Carolina, , Greensboro, North Carolina, 27402;3. Department of Biochemistry and Biophysics, University of Rochester Medical Center, , Rochester, New York, 14642;4. Computational Biology Center, IBM Thomas J. Watson Research Center, , Yorktown Heights, New York, 10598 |
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Abstract: | The global fold of human cannabinoid type 2 (CB2) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state 13C‐ and 15N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly 13C‐ and 15N‐labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. 13C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cβ, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the 13C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional 13Cα(i)? 13C?O(i) and 13C?O(i)? 15NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc. |
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Keywords: | cannabinoid receptor CB2 GPCR G protein‐coupled receptor solid‐state NMR molecular dynamics simulation |
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