To examine the structure and function of CXCR1 in its natural environment, we reconstituted the full-length, active receptor in phospholipid bilayers (proteoliposomes). The NMR method we developed, rotationally aligned (RA) solid-state NMR15
, is specifically tailored for the unique properties of membrane proteins in liquid crystalline phospholipid bilayers. It combines features of magic angle spinning (MAS)16
and oriented sample (OS)17
solid-state NMR to resolve and assign resonances associated with each amino acid residue, measure site-specific orientation restraints relative to the bilayer, and calculate the three-dimensional structure of the protein and its integral membrane orientation. It differs from previously used OS methods because it relies on the inherent rotational diffusion of membrane proteins in phospholipid bilayers18
to provide orientation-dependent motional averaging of dipolar coupling (DC) powder patterns relative to the bilayer normal rather than the orientation-dependent frequencies of single-line resonances observed in OS NMR of stationary, uniaxially aligned samples. Furthermore, the method takes advantage of recent bioinformatics developments that facilitate molecular fragment replacement approaches to structure determination, including membrane proteins19-21
CXCR1 was uniformly 13
N labeled by expression in Escherichia coli
, then purified, refolded in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) proteoliposomes22,23
, and placed as a concentrated suspension in a MAS rotor. Refolded CXCR1 binds IL-8 with high affinity (dissociation constant, Kd ~ 1–5 nM) and couples to its G-protein, Gi/o
(half maximum effective concentration, EC50
~ 1 nM)22
, indicating that the NMR sample conditions are compatible with physiological activity (Supplementary Fig. 1
As expected, the two-dimensional 13
C correlation spectrum of this 350-residue protein is quite crowded (Supplementary Fig. 2
). However, expansion of the 13
CA resonance region has sufficient resolution to contribute to the assignment process (Supplementary Fig. 2d
). Spectra, obtained from a uniformly 13
C labeled sample with a shorter mixing time and from a sample labeled using 2-13
C-glycerol, have fewer signals and improved resolution. Regardless, the vast majority of data used to resolve, assign and measure isotropic chemical shift frequencies from N, CA, CO, and CB sites were obtained from 13
C-detected three-dimensional, triple-resonance experiments (; Supplementary Figs. 3-4; Supplementary Table 2
). Overall, 97% of the backbone resonances for residues 20 to 325 were assigned. The missing resonances are from seven Pro (P22, P93, P170, P180, P185, P214, P257) and one Arg (R285). None of the 15
N and 13
C signals from the mobile N-and C-termini (residues 1-19 and 326-350) could be detected in the spectra, consistent with our observation of these signals in solid-state NMR experiments designed to detect only signals from mobile sites (Supplementary Fig. 6
), as well as our previous analysis of local and global motions of CXCR124
Structure determination of CXCR1
C-detected separated local field (SLF) experiments15
were used to measure the 1
N DC and 1
CA DC frequencies that provide orientation restraints for structure determination (; Supplementary Fig. 5
). The protein backbone structure was calculated by a molecular fragment replacement approach. An initial structural model was generated from a set of molecular fragments generated with CS-Rosetta20
from the experimental chemical shifts, the amino acid sequence of CXCR1, and the helical framework of the prototypical class A GPCR bovine rhodopsin25
. This initial model was first refined with the experimental restraints using the all atom19
and the implicit membrane21
potentials of Rosetta. Finally, the resulting structural model was refined by restrained simulated annealing using Xplor-NIH26
The three-dimensional structure of CXCR1 () has the consensus fold of a GPCR, with seven transmembrane helices (TM1-TM7) connected by three extracellular loops (ECL1-ECL3) and three intracellular loops (ICL1-ICL3). The average backbone pairwise RMSD is 1.7 Å (Supplementary Table 1
) and the experimentally measured 1
N DC and 1
CA DC values correlate remarkably well with those calculated from the refined protein structure (; Supplementary Fig. 7
). Notably, the correlations improve dramatically following refinement of the initial structural model with the experimental data, demonstrating that the NMR structure of CXCR1 is determined by the experimentally measured backbone orientation restraints and dihedral angles. We anticipate that both structural accuracy and precision will improve with inclusion of side chain restraints and, where feasible, distance restraints.
DCs contain information about molecular orientation as well as dynamics, and scaling of their values by local internal motions would compromise their analysis in terms of pure orientation restraints. For CXCR1, analysis of the DC data yielded similar values of the magnitude and symmetry of the molecular order tensor for residues in the helices and loops, indicating that these regions of the protein experience a similar degree of order in the lipid bilayer. This is supported by the excellent fit of the DC correlation plots obtained with a single value of the order tensor (; Supplementary Fig. 7b
) and by the observation that >20% of the 1
N DC signals from residues distributed throughout the protein sequence, including in loop sites, fall within 10% of the maximum value (21 kHz) expected for a static crystalline sample ().
Following the structure determination of rhodopsin from three- and two-dimensional crystals25,27
, the structures of several class A ligand-activated GPCRs have recently been determined by X-ray crystallography6,7
. CXCR1 is now the first GPCR with its structure determined in liquid crystalline phospholipid bilayers, and the first ligand-activated GPCR with its structure determined without modification of its amino acid sequence. The structure of CXCR1 shares significant similarities with that of CXCR428
, the only other chemokine receptor whose structure has been determined (; Supplementary Fig. 8
). However, there are some notable differences reflecting the modifications made to the sequence of CXCR4 required for crystallization (insertion of T4 lysozyme in ICL3, removal of 33 C-terminal residues and L125W mutation), the amino acid sequence differences between the two proteins, the influence of the planar phospholipid bilayer, or the rotational diffusion of the protein.
Structural comparison of CXCR1 (cyan, PDB ID: 2LNL) and CXCR4 (pink, PDB ID: 3ODU)
The CXCR1 helices are well defined by the spectroscopic data. For example, a plot of the measured values of the amide 1
N DC versus residue number yields a characteristic wave-like pattern29
reflecting helical structure (), with breaks in the waves corresponding to helix termini or kinks. For example, the DC data show the presence of a kink that changes the direction of helix TM2 (residues 74-101) at Phe88, coinciding with a kink at the same location in CXCR4 (). The extracellular start of TM7, just following ECL3, is tilted towards the receptor’s central axis in CXCR1, although it is less well defined in CXCR1 than in CXCR4 where residues in the N-terminus of TM7 interact with the added compound IT1t28
. Helix TM7 is also about one turn longer at the intracellular end than its counterpart in CXCR4, extending to Ile308, three residues beyond the conserved GPCR sequence NPxxY. Furthermore, residues immediately preceding the mobile C terminus of CXCR1 form a well-defined helix (H8; residues Gln310 to Ala321) that is absent in the structure of CXCR4 (, ; Supplementary Fig. 8
). H8 has a distinctly amphipathic amino acid sequence and aligns along the membrane surface, indicating that the phospholipid bilayer may play a role in stabilizing its conformation.
Three-dimensional structure of CXCR1
The NMR data further reveal the presence of two disulfide bonds (; Supplementary Fig. 9
) that are also present in CXCR4, one connecting the N-terminus to the extracellular start of TM7 (Cys30-Cys277) and the other connecting the extracellular end of TM3 to ECL2 (Cys110-Cys187). These Cys pairs are highly conserved in the sequences of chemokine receptors and are important for ligand binding. Together, they play a significant role in shaping the extracellular structure of the receptor, and also provide useful restraints for structure determination. The long ECL2 of CXCR1 forms a β-hairpin whose structure is constrained by the Cys110-Cys187 disulfide bond. A similar structure is observed in CXCR4 and in many other GPCRs, despite a lack of amino acid sequence similarity in this region. However, the ECL2 β-hairpin of CXCR1 is less well defined than that of CXCR4, consistent with the presence of two Pro residues in CXCR1.
In both CXCR1 and CXCR4, charged residues are mainly located near the membrane-water interface (Supplementary Fig. 10
), with negative charges clustered in the extracellular loops where they can play a role in ligand binding and receptor activation. In addition, four charged residues, contributed by helices TM2 (Asp85), TM3 (Lys117) and TM7 (Asp288, Glu291), form a polar cluster in the core of the helical bundle of CXCR1 that may have important consequences for ligand binding and receptor signal transduction. One of these residues (Asp288) is not conserved in CXCR4 and may contribute to the differences in biological activities of the two chemokine receptors.
The intracellular loops of GPCRs are critical for G-protein interactions11
. Modification of ICL3 by insertion of T4 lysozyme between TM5 and TM6, rendered CXCR4 incapable of activating G-proteins28
. In contrast, unmodified CXCR1 is fully active with respect to both G-protein activation and chemokine binding, and its three intracellular loops are structurally well defined (, , Supplementary Fig. 8
). Notably, ICL3, which is important for CXCR1 coupling to G-proteins and involved in calcium mobilization, chemokine-mediated migration, and cell adhesion, extends from Thr228 to Gln236, connecting helices TM5 and TM6, which are both one turn shorter than the corresponding helices in CXCR4. ICL3 protrudes into the cytoplasm where it is available for G-protein binding. Its sequence shares significant homology with other GPCRs; therefore, the ability to observe the structure of an intact GPCR provides an opportunity to propose structure-based mechanisms of G-protein binding and activation.
CXCR1 has been the subject of significant molecular modeling efforts aimed at understanding its interactions with small molecule inhibitors, including compounds active in reducing breast cancer metastasis30
. The structure of CXCR1 determined in a lipid bilayer membrane should facilitate these studies. The solid-state NMR approach used for structure determination has several significant advantages. The protein resides in fully hydrated liquid crystalline phospholipid bilayers at physiological conditions of temperature and pH, and no detergents or non-native lipid phases are present. The protein sequence is unmodified, with no truncations, mutations, or insertions of foreign proteins. The phospholipid composition can be varied, and other components, such as cholesterol, can be added. Other proteins or small molecules, including chemokines, G-proteins, nanobodies, and drugs can be added directly to the samples, enabling detection of any structural changes by direct spectroscopic and structural comparisons. NMR is adept at describing both overall and local protein dynamics. Measurements of DCs and isotropic chemical shifts to provide molecular orientation and dihedral angle restraints, combined with refinement in a membrane environment, facilitate structure determination by molecular fragment replacement. These can be supplemented with orientation restraints derived from rotationally averaged chemical shift anisotropy powder patterns to further improve structural accuracy and precision. Thus, we anticipate that this method will enable structure determination and structure-activity studies of other GPCRs as well as a wide range of other membrane proteins under near-native conditions.