The global fold of human cannabinoid type 2 (CB₂ ) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state ¹³ C- and ¹⁵ N 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 ¹³ C-, and ¹⁵ N-labeled CB₂receptor 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 C_α , C_β , 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 ¹³ C chemical shift distribution of C_α 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 C_α 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 ¹³ C_α (i)-¹³ C=O(i) and ¹³ C=O(i)-¹⁵ NH(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 2013;. © 2013 Wiley Periodicals, Inc.
Copyright © 2013 Wiley Periodicals, Inc., a Wiley company.

PMID:

 

23999926

 

[PubMed – as supplied by publisher]