title: Global fold of human cannabinoid type 2 receptor probed by solid-state 13C-, 15N-MAS NMR and molecular dynamics simulations.
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 (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 CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. (13)C 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 (13) C 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 (13) Cα(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.
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, Bethesda, Maryland, 20892.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kimura
authors.forename: Krishna
authors.initials: K
authors.lastname: Vukoti
authors.forename: Diane L
authors.initials: DL
authors.lastname: Lynch
authors.forename: Dow P
authors.initials: DP
authors.lastname: Hurst
authors.forename: Alan
authors.initials: A
authors.lastname: Grossfield
authors.forename: Michael C
authors.initials: MC
authors.lastname: Pitman
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Reggio
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Yeliseev
authors.forename: Klaus
authors.initials: K
authors.lastname: Gawrisch
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D008081:Liposomes; D056004:Molecular Dynamics Simulation; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D008081:Liposomes; D009587:Nitrogen Isotopes; D043885:Receptor, Cannabinoid, CB2; D011994:Recombinant Proteins
keywords: CB2; G protein-coupled receptor; GPCR; cannabinoid receptor; molecular dynamics simulation; solid-state NMR