Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles

Biophys J. 2009 Apr 22;96(8):3187-96. doi: 10.1016/j.bpj.2009.01.012.


The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for alpha-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [(15)N], [(15)N, (13)C], [(15)N, (13)C, (2)H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three alpha-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg(58) site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 +/- 0.10 A, 0.40 +/- 0.13 A, and 0.57 +/- 0.19 A, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G(56)VRSG(60) region. (15)N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Glycerides
  • Ligands
  • Micelles
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Conformation*
  • Receptors, Mating Factor / chemistry*
  • Receptors, Mating Factor / metabolism
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism


  • 1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-RAC-(1-glycerol))
  • Glycerides
  • Ligands
  • Micelles
  • Peptide Fragments
  • Receptors, Mating Factor
  • STE2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins