NMR Studies in Dodecylphosphocholine of a Fragment Containing the Seventh Transmembrane Helix of a G-Protein-Coupled Receptor from Saccharomyces cerevisiae

Abstract

The structure and dynamics of a large segment of Ste2p, the G-protein-coupled alpha-factor receptor from yeast, were studied in dodecylphosphocholine (DPC) micelles using solution NMR spectroscopy. We investigated the 73-residue peptide EL3-TM7-CT40 consisting of the third extracellular loop 3 (EL3), the seventh transmembrane helix (TM7), and 40 residues from the cytosolic C-terminal domain (CT40). The structure reveals the presence of an alpha-helix in the segment encompassing residues 10-30, which is perturbed around the internal Pro-24 residue. Root mean-square deviation values of individually superimposed helical segments 10-20 and 25-30 were 0.91 +/- 0.33 A and 0.76 +/- 0.37 A, respectively. 15N-relaxation and residual dipolar coupling data support a rather stable fold for the TM7 part of EL3-TM7-CT40, whereas the EL3 and CT40 segments are more flexible. Spin-label data indicate that the TM7 helix integrates into DPC micelles but is flexible around the internal Pro-24 site, exposing residues 22-26 to solution and reveal a second site of interaction with the micelle within a region comprising residues 43-58, which forms part of a less well-defined nascent helix. These findings are discussed in light of previous studies in organic-aqueous solvent systems.

Identification of Specific Transmembrane Residues and Ligand-Induced Interface Changes Involved In Homo-Dimer Formation of a Yeast G Protein-Coupled Receptor

“…Proline residues disrupt hydrogen bonding and have been shown to twist the standard structure of helices by introducing a kink between the segments contiguous with each other and to form molecular hinges (41, 63). The dynamic nature of the residues at the carboxyl side of Pro290 is consistent with the results of a high resolution analysis of a 73 residue fragment of Ste2p in DPC micelles which indicated that the Pro290 resulted in a kinked helix in this membrane mimetic environment (64). An irregular helical structure in part of a TM has been documented in the crystal structures of other GPCRs.…”

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

“…However, in the regions proximal to polar residues in the TMs (E and D in TM1 and TM2, respectively) the helices are destabilized, as judged by the reduction in the heteronuclear NOEs, by the TALOS predictions, by enhanced amide proton exchange and by the absence of contacts between sequential amide protons. Buried glutamic acid and aspartic acid residues are rarely found in TM domains of integral membrane proteins, and we have noted such increased flexibility on another isolated TM domain in DPC micelles (Neumoin et al 2007). The biological significance of these findings will be subject to future work.…”

“…A large body of literature supports the basic assumption of the model: For example, proteolysis of membrane proteins resulted in fragments containing entire TM sequences (Huang et al 1981), and chemically or recombinantly synthesized TM peptides spontaneously assembled thereby rescuing receptor activity (Kahn and Engelman 1992;Ridge et al 1995;Martin et al 1999;Wrubel et al 1994). Finally, peptides corresponding to the N and C terminus (Harmar 2001;O'Hara et al 1993), loop domains (Bennett et al 2004;Katragadda et al 2001a, b;Yeagle et al 2000) and transmembrane domains (Katragadda et al 2001a, b;Cohen et al 2008;Zheng et al 2006;Musial-Siwek et al 2008;Tian et al 2007;Lau et al 2008;Mobley et al 2007;Neumoin et al 2007) from GPCRs have been found to fold to distinct secondary structures which in certain cases resembled the structures of the corresponding regions of the intact receptor.…”

Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

“…Previous solution NMR-based studies of actual G protein-coupled receptors have most often involved fragments designed to mimic the conformation of the corresponding segments within the intact receptor [95, 398-403]. The tendency of some receptor sub-domains to adopt stable secondary or even tertiary structure can make characterization of these domains and their ligand binding properties a feasible alternative to studies of the intact receptor [400, 404-406].…”