Structure determination of a double transmembrane fragment of the G protein-coupled receptor Ste2p in membrane mimetic environments

G-protein coupled receptors (GPCRs) are relevant in cellular signal transduction pathways and are targets for disease therapeutics. Since the sequencing of the human genome, there have been close to 1000 GPCRs predicted and many have been characterized by biological and biochemical analysis. Though these integral membrane proteins (IMPs) have little sequence similarity, they show strong putative structural similarities. All GPCRs contain an N-terminal extracellular domain (NT), 7 transmembrane helical regions (TM) connected by intra- and extracellular loops (IL and EL, respectively), and a C-terminal intracellular tail (CT). The extracellular domains are thought to play a role in ligand-receptor interactions and together with the TM domains form the ligand binding site for many GPCRs.;The number of structures in the protein structural database is increasing exponentially, but there are only a few high-resolution GPCR structures, those of rhodopsin, the beta-adrenergic receptors and the adenosine A2A receptor. GPCRs are difficult to crystallize and the protein:detergent complex size can be restrictive for solution NMR. As is true of GPCRs in general, limited structural information is available for Ste2p, a yeast GPCR which recognizes the alpha-factor tridecapeptide mating pheromone. Many groups, including the Naider lab, have been working with smaller fragments of GPCRs in an attempt to elucidate the structure of individual regions by either crystallization or NMR and to combine these to achieve a better picture of GPCR structure. The use of fragments of GPCRs to study the structure of these large membrane receptors remains controversial. I have used Ste2p as a paradigm to answer the following questions: (1) Can GPCR fragments fold into a tertiary structure without the context of the full protein? and (2) Do organic:aqueous solvents such as trifluoroethanol(TFE):water result in GPCR fragment structures similar to those found in detergents and lipids?;I have cloned and expressed two 2TM regions of Ste2p, G31-T110 (TM1-TM2) and R231-S339 (TM6-TM7-CT40). The constructs were chosen based on the biological relevance of the domain and a buried surface area analysis that was performed using a rhodopsin-templated model of Ste2p and the NACCESS software package. Expression in Escherichia coli in minimal media and CNBr cleavage conditions for these 2TM fusion proteins were optimized and the fusion protein was labeled with [15N], [15N, 13C], [15N,13C,2H] and with selectively [15N]-labeled amino acids to aid in NMR assignments. Purification of isotopically labeled peptides was performed using RP-HPLC with a Zorbax C3 column and an acetonitrile:water gradient containing isopropanol and 0.1% TFA. Yields of Ste2p(G31-T110) were 6-20 mg per L culture. Far UV CD analysis indicated that Ste2p(G31-T110) formed a highly helical peptide in TFE:water and various micellar environments, whereas the helicity was reduced for Ste2p(R231-S339). Initial [15N,1H]-HSQC analysis showed that CD can be used as an NMR screening technique to determine conditions for high resolution NMR analysis.;Ste2p(G31-T110) was used to determine a high-resolution structure of a 2TM GPCR fragment in TFE:water(+0.1% TFA) (1:1; v/v) by multidimensional NMR spectroscopy. The 80-residue polypeptide was folded into a helical hairpin, but the overall convergence of the structure bundle was poor with an RMSD of ~ 6.7 A. Comparison to an NMR study of TM1-TM2 in LPPG micelles concluded that very different structures occurred in the two membrane mimetic media and it was observed that relative conformation of TM2 to TM1 in TFE:water was twisted in comparison to that in the LPPG micelle so that the interactions between helices were on opposite sides. Although Ste2p(G31-T110) folds into a tertiary structure in TFE:water (1:1; v/v) the structure is quite flexible compared to that in LPPG micelles.