Global fold of human cannabinoid type 2 receptor probed by solid-state super(13)C-, super(15)N-MAS NMR and molecular dynamics simulations

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2014-03, Vol.82 (3), p.452-465
• Main Authors: Kimura, Tomohiro, Vukoti, Krishna, Lynch, Diane L, Hurst, Dow P, Grossfield, Alan, Pitman, Michael C, Reggio, Patricia H, Yeliseev, Alexei A, Gawrisch, Klaus
• Format: Article
• Language: English
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.24411

The global fold of human cannabinoid type 2 (CB sub(2)) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state super(13)C- and super(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 super(13)C- and super(15)N-labeled CB sub(2) receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. super(13)C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C sub([alpha]), C sub([beta]), 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 super(13)C chemical shift distribution of C sub([alpha]) resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB sub(2). Thus the shape of the C sub([alpha]) band can be used as an indicator of CB sub(2) 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 ( greater than or equal to 1.5 ppm for carbons and greater than or equal to 5.0 ppm for nitrogens). Simulated two-dimensional super(13)C sub([alpha])(i)-- super(13)C[double horizonal line]O(i) and super(13)C[double horizonal line]O(i)-- super(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 CB sub(2) by solid-state MAS NMR. Proteins 2014; 82:452-465. copyright 2013 Wiley Periodicals, Inc.