Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy

Abstract

Seven-helix membrane proteins represent a challenge for structural biology. Here we report the first NMR structure determination of a detergent-solubilized seven-helix transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II (pSRII) from Natronomonas pharaonis, as a proof of principle. The overall quality of the structure ensemble is good (backbone r.m.s. deviation of 0.48 A) and agrees well with previously determined X-ray structures. Furthermore, measurements in more native-like small phospholipid bicelles indicate that the protein structure is the same as in detergent micelles, suggesting that environment-specific effects are minimal when using mild detergents. We use our case study as a platform to discuss the feasibility of similar solution NMR studies for other 7TM proteins, including members of the family of G protein-coupled receptors.

Figure 1

Side chain assignment and NOE-derived distance restraints in pSRII. (a) Extent of the side chain assignments mapped onto a schematic topology cartoon of pSRII displaying the α-helices A–G and loops L1–L6. Residue types are color coded as Ile, Leu, Val in red, Ala, Met, Thr in blue, Phe, Trp, Tyr in yellow and all remaining residues in green. Residues with full proton assignments are encircled in bold while no circles indicate only partial side chain assignments. (b) A combination of ¹³C-¹³C cross-sections at the methyl ¹H frequencies of representative Ile, Leu and Val residues from methyl-separated experiments (left) and ¹H-¹³C strip plots of the same residues at the corresponding backbone ¹⁵N frequencies (right) from 3D J-assignment experiments shows the correlation of backbone signals with the methyl resonances. (c) ¹H-¹H cross-sections from 3D ¹⁵N- or ¹³C-separated NOESY-HSQC spectra at the ¹⁵N (blue) or ¹³C (green) frequencies of selected residues. Intra- and inter-residue NOEs are indicated with annotations.

Figure 2

Solution-NMR structure of pSRII in DHPC micelles. (a) An ensemble of 30 low-energy structures derived from NMR restraints. The backbone r.m.s. deviation for residues 1–221 is 0.48 Å. Loop and strand regions are indicated in green and purple, respectively. The 20 unstructured C-terminal residues are omitted from this figure. (b) Ribbon diagram of the structure closest to the mean. (c) Superposition of the NMR structure shown in b in red and the X-ray crystal structure 1H68 in blue. The r.m.s. deviation of the best backbone superposition of the two structures for residues 1–219 is 1.23 Å. (d) All-trans retinal binding pocket viewed side-on showing the chromophore attached via a Schiff base to Lys205. The closest to the mean NMR structure is shown in pink superimposed on the X-ray structure displayed in blue. Selected side chains of residues contacting the retinal are shown for the NMR structure in purple. The two potential counter ions to the Schiff base Asp75 and Asp201 as well as Arg72 are displayed in brown.

Figure 3

Mapping of hydrophobic and solvent exposed regions of pSRII through titration with the detergent spin labels 16-DSA and gadoteridol. (a) Residues that experience a significant relaxation enhancement mapped onto the NMR structure of pSRII. Residues that interact with 16-DSA with a relaxation enhancement ε > 35 mM⁻¹s⁻¹ are shown in purple. They are in close contact to the hydrophobic interior of the micelle. Solvent exposed residues that interact with the water-soluble gadoteridol resulting in an enhancement ε > 4 mM⁻¹s⁻¹ are shown in green. These residues are in the vicinity of the polar detergent head groups possibly in contact with water. Residues represented in light grey are unaffected, with ε values below these thresholds. (b) Relaxation enhancement values obtained from the titration with the two spin labels. The same color scheme is employed as in a. A graphical representation of the secondary structure is displayed at the top of the diagram.

Figure 4

Comparison of small bicelle versus micelle reconstitution of pSRII. (a) Overlay of [¹H,¹⁵N]-TROSY correlation 2D spectra showing the backbone amide resonances of pSRII in DHPC micelles (cyan) and DMPC/c6-DHPC (q=0.3) small bicelles (red). (b) Chemical shift changes Δδ(¹H,¹⁵N) = [(Δδ¹H)² + (Δδ ¹⁵N)²/6]^0.5 in a are mapped onto the structure of pSRII. Increasingly larger differences are indicated by a gradual color change from blue to red as shown in the figure. The differences between micelle and small bicelle environments are generally small. Affected residues are located on the exterior of the protein mainly towards the helix extremities. (c) Overlay of ¹H-¹H cross-sections from 3D ¹⁵N-separated NOESY-HSQC spectra taken at the ¹⁵N frequencies of residues 97–101 for pSRII solubilized in either a micelle (blue) or small-bicelle (red) environment. Despite the chemical shift changes observed for these residues, the cross-peak pattern is unchanged indicating the preservation of the structural features. Sequential NOEs are annotated. The larger line width in the small bicelle spectra is due to the slower tumbling of the protein–bicelle complex.