Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

Abstract

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.

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

“…These conditions were similar to those previously used in our investigation of TM12 (37). The NMR samples of TM1, TM123 and TM127 all exhibit homogeneous line-widths and good signal dispersion considering their highly alpha-helical nature (see Fig.…”

“…7A, lane 7); in fact, a protein band corresponding to doubly-glycosylated forms became predominant whilst triplyglycosylated forms were not observed. These results suggest that TM12 is being inserted into the membrane as a helical hairpin (26,37), with both N-and C-termini oriented toward the ER lumen (Fig. 7B, central panel).…”

“…For TM1 97% of the backbone and 66% of the side chain assignments were achieved. The assignments for TM12 as well as details of its structure calculation have been published previously (37).…”

“…In all fragments cysteine and methionine residues were replaced by amino acids with similar properties (Ser for Cys; Ile, Leu and Val for Met residues) to retain feasibility of chemical cleavage and preserve functionality of the receptor as reported before (34,35 (37). To obtain additional long-range distance restraints for TM127, the single cysteine mutants S47C, S75C and S104C containing the full N-terminus (S2-T114, T274-L340), and an N-terminal His 10 -tag separated by a cloning insertion coding for a C3 site (His 10 -Leu-Glu-Val-Leu-Phe-Gln-GlyPro-Ser2….)…”

“…This resulted in expression yields of about 15mg and 5mg of purified protein per liter of 15 N 13 C-labeled and 15 N 13 C 2 H-labeled M9 culture, respectively, for TM1 (using the strain BL21-AI), 11mg/L and 5mg/L for TM123 (BL21-AI) (36) and 15mg/L and 5mg/L for TM127 (BL21(DE3)STARpLysS). Expression details of TM12 have been reported previously (37). To obtain additional long-range distance restraints for TM127, the single cysteine mutants S47C, S75C and S104C containing the full N-terminus (S2-T114, T274-L340), and an N-terminal His 10 -tag separated by a cloning insertion coding for a C3 site (His 10 -Leu-Glu-Val-Leu-Phe-Gln-GlyPro-Ser2….)…”