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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Biol. Author manuscript; available in PMC 2012 November 25.
Published in final edited form as:
PMCID: PMC3236022

Interactions of Interleukin-8 with the Human Chemokine Receptor CXCR1 in Phospholipid Bilayers by NMR Spectroscopy


CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity, and subsequently transduces a signal across the membrane bilayer to a G-protein activated second messenger system. Here we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly 15N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the receptor-containing bilayers immobilizes IL-8 (on the ~105 Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the receptor in phospholipid bilayers, and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enable us to propose a model for a multi-step binding process.

Keywords: membrane protein, solid-state NMR, lipid bilayer, CXCR1, IL-8


The chemokine system regulates many biological and pathological processes, including inflammation, embryogenesis, metastasis, host defense against infection, and innate immunity.1 Its broad role in regulation is accomplished through the binding of specific chemokines to their respective G-protein coupled receptors (GPCRs). For example, the release of the chemokine interleukin-8 (IL-8) by several cell types is a response to an inflammatory stimulus, and results in the migration of leukocytes, including neutrophils, monocytes, T- and B-lymphocytes and basophils, to these sites. IL-8 has also been shown to stimulate self-renewal of breast cancer stem cells in vitro.2 In humans, two high affinity IL-8 receptors, CXCR1 and CXCR2, have been characterized,3; 4 and CXCR1 has been identified as a target for blocking the formation of breast cancer stem cells that drive tumor growth and metastasis.5

CXCR1 belongs to the family of chemokine receptors with seven trans membrane helices that couple to heterotrimeric G-proteins for signal transduction.6 We have demonstrated the expression in E. coli, and purification and refolding of functional full-length CXCR1, and numerous constructs of the receptor, including N-terminal truncated CXCR1 (NT39-350), C-terminal truncated CXCR1 (CT1-319), both N- and C-terminal double truncated CXCR1 (DT23-319), the first trans membrane helix domain of CXCR1 (1TM1-72), and the N-terminal extracellular domain (ND1-38) without any residues associated with the first trans membrane helix.7; 8 We have also characterized the local and global dynamics of full-length CXCR1 in membrane environments using a combination of solution NMR and solid-state NMR techniques.9

The mechanisms by which chemokines modulate specific biological activities are central to understanding how GPCRs transmit signals through the membrane bilayer to the interior of the cell. Previously, solution NMR spectroscopy has been used to characterize the structure of IL-8 alone,10; 11; 12 and bound to synthetic peptides with sequences corresponding to portions of the N-terminal domain of CXCR1.12; 13 Solution NMR is feasible in these situations because of the small size and high solubility of IL-8 and the peptides derived from the N-terminal sequence of CXCR1. These studies have identified a probable location on IL-8 that interacts with the N-terminal domain of CXCR1; however, these model systems lack several essential components of the biological system, namely the additional residues present in full-length GPCR and the planar lipid bilayer environment where the receptor resides. For example, the earlier studies using relatively short synthetic peptides could not detect interactions with extracellular loops or other regions of CXCR1 or the effects of lipid bilayers on the structures, dynamics, and interactions of CXCR1 and IL-8.

Here we describe studies that use uniformly 15N-labeled full-length CXCR1, several of its truncated constructs, two versions of its N-terminal domain, and native IL-8 in both free and bound states. By utilizing both solution NMR and solid-state NMR experiments it was possible to monitor the proteins in a wide range of lipid environments, including phospholipid bilayers. Appropriate control experiments on both of the proteins in the various lipid environments enable us to propose a multi-step model for the interactions between IL-8 and CXCR1 in lipid bilayers.


Interaction of the N-terminal domain of CXCR1 with membranes

We expressed, purified, and characterized the N-terminal extracellular domain of CXCR1 (ND1-38) that corresponds to the first 38 residues of CXCR1. The 15N chemical shift OS solid-state NMR spectrum of uniformly 15N-labled ND1-38 in magnetically aligned bilayers demonstrates that the sample is well aligned on the surface of the bilayers (Fig. 1a). The signals that result from cross-polarization are neither centered at the isotropic frequency nor have the appearance of a powder pattern, providing strong evidence for the existence of specific interactions between the phospholipids and amino acid residues in the N-terminal domain of CXCR1. As a control, IL-8 was subject to cross-polarization in the presence of magnetically aligned bilayers not containing a construct of CXCR1, and as expected, no NMR signals were observed (data not shown) because IL-8 is water-soluble and does not interact with phospholipids.

Fig. 1
Membrane interaction of ND1-38 and dissociation of the ND1-38-IL-8 complex from the membrane. (a and b) 15N chemical shift OS solid-state NMR spectrum of uniformly 15N-labeled ND1-38 alone (a) and in complex (b) with unlabeled IL-8 in q=3.2 bicelles. ...

The 1H-15N HSQC solution NMR spectrum of ND1-38 in aqueous buffer (Fig. 1b, black contours) has a very limited dispersion of 1H amide chemical shifts (< 1 ppm), which is typical of relatively small peptides with little or no secondary or tertiary structure. Moreover, no homonuclear 1H/1H NOE cross-peaks could be observed in standard two-dimensional experiments. In contrast, IL-8 yields a well-resolved solution NMR spectrum that is typical of a native globular protein, since it has a wide dispersion (> 6 ppm) of 1H amide chemical shifts, and relatively narrow line widths (Fig. S1b).

Compared to aqueous solution, there are significant chemical shift changes and broadening of a subset of backbone amide signals of ND1-38 including the side chain signal of Trp10 when lipids are added to the sample (Fig. 1c and Fig. S1a). In contrast, IL-8 does not interact with lipid bilayers and therefore no significant spectral changes including to the side chain signal of Trp57 were observed in the presence of phospholipid bilayers (Fig. S1b). This is consistent with the OS solid-state NMR result on IL-8 alone in the presence of lipid bilayers.

The samples made from mixtures of long chain phospholipids (e.g. DMPC) and short chain phospholipids (e.g. DHPC), have their molar ratio (long/short) characterized by the parameter “q” and are referred to as “bicelles”. These protein-containing lipid mixtures enable the structures and dynamics of the proteins to be characterized by solution NMR and solid-state NMR experiments; q values less than about 1.5 result in isotropic bicelles that are generally suitable for solution NMR experiments, and those with values greater than about 2.5 form magnetically alignable bilayers that immobilize the protein and require solid-state NMR methods to obtain high-resolution spectra.14; 15; 16; 17

In isotropic q=0.1 bicelles, the largest chemical shift changes were observed primarily near the N-terminus (residues 2-16) of ND1-38 (Fig. S1a). In magnetically aligned q=3.2 bilayer samples, the most affected signals, including that from the Trp10 side chain, were broadened beyond detection in solution NMR spectra (Fig. 1c, red contours). This significant broadening of the first 16 residues of CXCR1 does not result from weak alignment of the protein in the liquid crystalline phase but rather from the interactions with the lipid bilayers, since in a control experiment all the signals that were only slightly broadened could be observed when ND1-38 was weakly aligned using fd bacteriophage particles in aqueous buffer solution.

Dissociation of the N-terminal domain of CXCR1 bound to IL-8 from membranes

The spectra of IL-8 bound to ND1-38 in lipid bilayers provide insights into the ternary complex of IL-8, CXCR1, and phospholipid bilayers (Fig. 1b and d). There were no significant chemical shift changes in the solution NMR spectrum of the ND1-38 bound to IL-8 when lipid bilayers were added to the aqueous buffer. Remarkably, the signals of free ND1-38 that were broadened out due to the membrane interaction (Fig. 1c, red contours) reappear when IL-8 is bound to ND1-38, including the Trp10 side chain signal (Fig. 1d, red contours). Overall, the line widths of the signals from ND1-38 bound to IL-8 are only slightly broader than those of free ND1-38. Taken together these results demonstrate that ND1-38 does not interact with lipid bilayers when bound to IL-8, and IL-8 does not interact with bilayers in the absence of the N-terminal domain of CXCR1. The inability, despite extensive efforts, to obtain solid-state NMR signals from ND1-38 when it is complexed with IL-8 in the presence of aligned phospholipid bilayers further supports the finding that the binding of IL-8 results in the dissociation of the N-terminal domain of CXCR1 from phospholipid bilayers (Fig. 1b).

Binding site mapping of the IL-8 and CXCR1 complex

The backbone resonance assignments of free IL-8 under the experimental conditions used here were made by comparisons to the previously assigned spectra10 and confirmed by comparisons with 1H-15N HSQC spectra of selectively Leu, Ile, Val, and Phe 15N-labeled samples as well as conventional triple resonance experiments performed on uniformly 13C/15N labeled samples.

The amino acid residues that form the binding sites of IL-8 and of ND1-38 were identified by mapping the chemical shift perturbations resulting from complex formation between one uniformly 15N-labeled polypeptide in the presence of its unlabeled counterpart. The expanded region of 1H-15N HSQC solution NMR spectra of uniformly 15N-labeled IL-8 shows the specific chemical shift perturbation of backbone amide resonances following the addition of unlabeled ND1-38 (Fig. 2a). The plot of chemical shift changes as a function of residue number indicates that relatively large chemical shift changes (> 0.06 ppm) are observed in three distinct regions of the IL-8 sequence: residues 12, 17 and 20 in the N-loop; residues 44, 48, 49 and 50 in the third β-strand; and residues 61 and 62 in the C-terminal helix (Fig. 2d). This identifies the regions of IL-8 that interact with the N-terminal domain of CXCR1. These findings are similar to those from previous studies performed with a synthetic peptide corresponding to the first 40 residues of the N-terminal domain of CXCR118 and with a 17-residue peptide, corresponding to residues 9-29 of CXCR1 where residues 15-19 were replaced with a single six-amino hexanoic acid moiety.13

Fig. 2
Interaction of IL-8 with the truncated CXCR1 constructs. (a-c) Expanded region of 1H-15N HSQC solution NMR spectra: (a) Uniformly 15N-labeled IL-8 alone (black contours) and in complex with unlabeled ND1-38 (red contours) in aqueous buffer; (b) Uniformly ...

It has been reported that not only the N-terminal domain but also the extracellular loops of CXCR1 are involved in the interaction with IL-8.19 The spectral changes of IL-8 by the addition of N-terminal truncated CXCR1 (NT39-350) in q=0.1 isotropic bicelles provide evidence for the specific interactions between IL-8 and extracellular loops of CXCR1 (Fig. 2b). Although the extent of the chemical shift perturbations of IL-8 by NT39-350 was not as large as those by ND1-38, significant line broadening of the signals, except the first N-terminal 6 residues, and relatively large chemical shift changes of Leu17 and Lys23 of IL-8 were observed (Fig. 2e).

The binding site of the N-terminal region of CXCR1 has been characterized by the measurement and analysis of intermolecular NOEs observed between IL-8 and the 17-residue peptide derived from CXCR1 described above.13 Here, we take advantage of having prepared an isotopically labeled polypeptide by bacterial expression corresponding to the N-terminal domain of CXCR1 to map the binding site using heteronuclear solution NMR experiments. The changes in the spectrum of ND1-38 resulting from the addition of unlabeled IL-8 have the characteristics of “fast exchange” on the timescales of the 1H and 15N chemical shifts. With increasing concentrations of IL-8, the amide resonances of the affected residues shift incrementally from the frequencies observed in the free state to those of the fully bound state (Fig. 2c). The chemical shift frequencies stop changing when approximately one equivalent of the unlabeled IL-8 monomer has been added to the solution containing labeled ND1-38 (Fig. 2f). The binding affinity of ND1-38 and IL-8 was determined by treating the binding-induced chemical shift changes as a titration.20 The KD is approximately 70 μM under these conditions. Previously, N-terminal fragments of CXCR1 have been shown to bind IL-8 with an affinity three to five orders of magnitude weaker than that of the full-length receptor.13; 18

Binding of IL-8 to full-length CXCR1 in membrane environments

Interactions of IL-8 with polypeptides whose sequences are derived from the N-terminal region of CXCR1 have been described previously.13; 18; 21 However, information about the interaction of IL-8 with full-length CXCR1 is scarce, largely because of the experimental difficulties encountered in the study of large membrane proteins in phospholipid bilayers. We have developed protocols for the expression, purification and refolding of various functional CXCR1 constructs in lipid bilayers including the full-length protein.7; 8 This enables us to study the interactions of IL-8 with full-length and truncated constructs of CXCR1 in membrane environments.

Fig. 3 shows the effects of increasing the amounts of CXCR1 in bilayers to a q=0.1 isotropic bicelle solution containing uniformly 15N-labeled IL-8. In the absence of the receptor-containing bilayers, the 15N edited 1H solution NMR spectrum of the amide region has narrow and well-dispersed resonances, typical of a small globular protein in aqueous solution. As the addition of the receptor approaches a 1:1 molar ratio of CXCR1:IL-8, nearly all signals from labeled IL-8 broaden systematically and disappear into the baseline, with the exception of a few signals that have been assigned to residues near the N- and C-termini. The result was more dramatic in lipid bilayers, because with CXCR1 in proteoliposomes at a 1:1 molar ratio with IL-8, all of the IL-8 signals disappear as a result of their immobilization upon binding to the CXCR1-containing bilayers. Refolded CXCR1 prepared by our methods has been shown to bind IL-8 with an affinity (KD 1 - 5 nM) and to couple to G-protein (EC50 ~ 1 nM)7; 8, which are similar to the values previously reported in the literature.3

Fig. 3
Interaction of IL-8 with full-length CXCR1. 15N edited 1H solution NMR spectra of uniformly 15N-labeled IL-8 in the presence of unlabeled full-length CXCR1 in q=0.1 isotropic bicelles. The molar ratios of CXCR1 to IL-8 monomer are listed on the right ...

Critical role of the N-terminal domain of CXCR1 for IL-8 binding

Comparisons of 15N chemical shift OS solid-state NMR spectra of uniformly 15N-labeled IL-8 bound to unlabeled full-length CXCR1, and constructs consisting of the first trans-membrane helix domain (1TM1-72) and the N-terminal truncated (NT39-350) receptors in lipid bilayers are shown in Fig. 4. These results demonstrate that the N-terminal domain of CXCR1 is mainly responsible for the binding of IL-8. The OS solid-state NMR signals of IL-8 were intense and well resolved when IL-8 was added to full-length and 1TM1-72 receptors aligned in lipid bilayers, demonstrating that their interaction is strong enough to immobilize and align IL-8 along with the receptor at a unique orientation in the magnetically aligned bilayers (Fig. 4a and b). As a control, no IL-8 signals could be observed in OS solid-state NMR experiments in a sample containing labeled IL-8 and an unlabeled NT39-350 (Fig. 4c). Since binding to the receptor is necessary to immobilize and align the IL-8, this suggests that the binding site is predominantly located in the N-terminal region of the receptor.

Fig. 4
Interaction of IL-8 with various CXCR1 constructs in lipid bilayers. 15N chemical shift OS solid-state NMR spectra of uniformly 15N-labeled IL-8 in complex with three different constructs of CXCR1 in q=3.2 aligned bicelles: (a) Full-length CXCR1; (b) ...


Comparisons between the solution NMR and solid-state NMR spectra of ND1-38 alone and bound to IL-8 provide information about the roles of the lipid bilayers on interactions of the N-terminal domain of CXCR1 and IL-8. The N-terminal region of CXCR1 determines the specificity and affinity for IL-8.22; 23 Recently, a 34-residue peptide with a sequence corresponding to the N-terminal residues of rabbit CXCR1 was shown to interact with the membrane surface by monitoring fluorescence of two tryptophan residues of the peptide.24 Both human and rabbit CXCR1 receptors have similar affinity and specificity for human IL-821 and their N-terminal domains have high sequence homology (Fig. S2). Tryptophan residues are commonly found near the membrane surface, since the polar amide group and hydrophobic ring structure of this amino acid facilitate its localization at the polar/apolar interface.25 Significantly, signals from both the backbone and the side chain of the tryptophan residue in ND1-38 are broadened beyond detection in the presence of lipid bilayers (Fig. 1c), suggesting that the tryptophan residue may serve as an anchor on the membrane surface. The tryptophan residues located in the N-terminal domain of rabbit CXCR1, one of which is located in the same position as a tryptophan in the human CXCR1 sequence, have also been shown to be involved directly in membrane interactions.24

The chemical shift perturbation plot for labeled IL-8 in Fig. 2d obtained by the addition of unlabeled ND1-38 shows substantial changes in three regions of the primary sequence. The residues that contribute to the binding cleft identified in the three-dimensional structure of IL-8 were the ones most strongly affected by the interaction with ND1-38. The central region of the ND1-38 primary sequence (residues 18-27) was most strongly affected by binding to IL-8. This suggests that ND1-38 may adopt an extended conformation when complexed to IL-8. Although the proline residues of ND1-38 were not monitored in our experiments, alanine-scanning studies have shown that the two prolines, 21 and 29, as well as Tyr27 contribute to the interactions with IL-8, suggesting that the hydrophobic characteristics of these residues play roles in binding to the N-terminal domain of CXCR1.26

Many studies of chemokines and their interactions with receptors have concluded that one or more of the extracellular loops of the receptors are involved. In particular, alanine-scanning experiments have shown that the third and fourth extracellular loops of CXCR1 are involved in the binding to IL-8.19 An overall broadening of solution NMR signals of IL-8 in the presence of 1TM1-72 (data not shown) and NT39-350 (Fig. 2b) at a molar ratio of 1:1 was observed, but in both cases the signals were less affected than those of IL-8 in the presence of the full-length receptor (Fig. 3). Two possible reasons for this difference are that the binding of IL-8 to 1TM1-72 is not as tight as for the full-length receptor, or that the binding is as tight as full-length receptor, but the smaller size of the IL-8 and 1TM1-72 complex (~ 18 kDa) reorients much faster than IL-8 and the full-length complex (~ 52 kDa) in isotropic q=0.1 bicelles. In the case of the N-terminal truncated receptor, the molecular mass of NT39-350 is reduced by only 10% compared to the full-length receptor; thus, the reduction in rotational correlation time is unlikely to be sufficient to account for the spectral changes. It may be that the changes are a manifestation of weak interactions of IL-8 to extracellular loop regions of the receptor without the contributions from the missing residues in the N-terminal domain of the receptor.

The role of dimerization of IL-8 in binding CXCR1 is not fully understood, but recent studies have shown that the IL-8 monomer binds to the N-terminal domain of CXCR1 with higher affinity than the IL-8 dimer.27; 28 We used only the monomeric form of CXCR1, and in all of our experiments the spectral changes stopped when an approximately equimolar concentration of CXCR1 monomer to the IL-8 monomer was achieved. These results suggest that one molecule of CXCR1 binds to one molecule of the IL-8 monomer. Since IL-8 exists as a stable homodimer in an aqueous solution, it is possible that the chemical shift perturbation of IL-8 upon binding to CXCR1 constructs results from not only the direct interaction between them but also from the dimer to monomer transition of IL-8.

It is essential to obtain atomic-resolution structural details about how IL-8 interacts with its high affinity membrane-embedded receptors in order to understand the first step of the complex signaling cascade. In the meantime, we interpret the NMR results discussed above in terms of a multi-step series of interactions between IL-8 and CXCR1 with significant contributions from the phospholipid bilayers (Fig. 5). Thus, we propose that the ternary complex of IL-8/CXCR1/bilayer is the essential species.

Fig. 5
Model of IL-8 interacting with CXCR1 in membranes. Step 1: N-terminal domain of CXCR1 (green) is flexible yet structured by interacting with the surface of the membrane, contributing to receptor selectivity. The first half of the domain is mainly involved ...

In the first step, the N-terminal domain of CXCR1, which has many characteristics of a peripheral membrane protein, interacts transiently with the membrane surface, and adopts a relatively well-defined yet flexible structure that may contribute to receptor selectivity. Our NMR data on the N-terminal domain of CXCR1 in the absence and presence of phospholipid bicelles clearly demonstrates the significant effects of the membrane environment on the structure and dynamics of this domain (Fig. 1). In particular, the Trp10 side chain is likely to be embedded in the bilayer.

In the second step, after binding to IL-8, the N-terminal domain dissociates from the membrane surface. Upon interaction with IL-8, the solution NMR signals of the N-terminal domain that were completely broadened out due to the membrane interaction (step 1) reappeared as a result of dissociation of the domain from the membrane (Fig. 1d). The complementary OS solid-state NMR spectrum of the domain in complex did not yield any signals, which also demonstrates that the complex is no longer immobilized by interactions with the membrane (Fig. 1b).

In the third step, the complex of IL-8 and the N-terminal domain rearranges to engage a second binding site on the receptor, most likely involving one or more extracellular loops (Fig. 2b and e). This step might be the trigger for the conformational changes in the receptor needed to activate secondary signaling cascades. This does not exclude the possibility that IL-8 interacts simultaneously with the N-terminal domain and extracellular loops of the receptor.

A two-site mechanism of chemokine-receptor interaction in which the N-terminal domain and extracellular loop in the receptor are involved in the ligand interaction has been proposed based on the various structure-function studies reviewed by Rajagopalan and Rajarathnam.29 Although it is not fully understood how the two-site mechanism mediates affinity, selectivity, and activation of the receptor, the N-terminal residues of the receptor are shown to be essential for both binding affinity and receptor selectivity.22 The OS solid-state NMR data presented here show that the N-terminal domain of CXCR1 is mainly responsible for the strong interaction with IL-8 (Fig. 4).

It has been proposed that the chemokine N-terminal “ELR” motif interacts with the extracellular loops of the receptor.30; 31 Recently, the highly dynamic N-terminus including the ELR motif of the chemokine SDF-1 has been proposed to play a crucial role in the interaction with its receptor CXCR4.32 However, we do not observe experimental NMR evidence that the N-terminal ELR motif of IL-8 interacts with full-length or N-terminal truncated CXCR1. This may be due to differences between the two receptors, or it may require future studies of the structures and mechanisms of G-protein coupled receptors to fully sort out.

The interactions between ligands and their membrane-embedded receptors, especially GPCRs, are the first step in initiating the complex cascades of protein interactions known to regulate physiological processes in mammals. Here we demonstrate that the interaction between IL-8 and its receptor, CXCR1, must be analyzed in the context of the phospholipid bilayer environment. Solid-state NMR spectroscopy is unique in providing atomic-resolution information about membrane proteins and their complexes in phospholipid bilayers under conditions where signal transduction occurs. The resulting NMR data enable us to propose a model for the interactions between IL-8 and CXCR1 that involve the phospholipid bilayer, IL-8, the N-terminal domain of CXCR1, and residues in inter-helical loops near the C-terminus. In summary, the membrane bilayer plays a role that is as important as the structural features of the two protein components in the interactions of IL-8 and CXCR1 in the first step of transducing biological signals.

Materials and Methods

Sample preparation

IL-8 was expressed and purified as described previously.22 Full-length CXCR1 and three truncated constructs including N-terminal truncated CXCR1 (NT39-350), the first trans membrane helix domain of CXCR1 (1TM1-72), and the N-terminal extracellular domain of CXCR1 (ND1-38) were expressed, purified and refolded as described previously.7; 8 The amino acid sequences of the CXCR1 constructs are shown in the supporting information. The amino acid sequence of ND1-38 substitutes Ser for Cys at position 30 to prevent complications due to inter-molecular disulfide bond formation.

For the solution NMR experiments, the concentration of IL-8 and ND1-38 polypeptides was 0.1 mM, in 20 mM HEPES, at pH 5.5, in 400 μl of 90% H2O/10% 2H2O. The protein-containing bicelle samples of IL-8 and ND1-38 were prepared by dissolving the lyophilized polypeptides directly into premixed solutions containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) phospholipids. The lipids were purchased from Avanti Polar Lipids ( The isotropic (q=0.1) and magnetically alignable (q=3.2) samples contain 10% DHPC (w/v) and 10% DMPC (w/v), respectively. The samples of the CXCR1 constructs, except for the soluble ND1-38 polypeptide, were prepared from proteoliposome pellets (20% wt lipid) in which 1 mg of the polypeptide was reconstituted into a solution containing 10 mg of DMPC. For the titration experiments, a stock solution of the unlabeled proteins in the same buffer conditions was added to the uniformly 15N-labeled proteins so that the final molar ratios were 0.25, 0.5 and 1.0.

For the OS solid-state NMR experiments, 1 mg of the unbound form of uniformly 15N-labeled ND1-38 and IL-8 were dissolved in 200 μl of a q=3.2 lipid mixture containing 20% DMPC (w/v), 20 mM HEPES, at pH 5.5. The complex was formed by adding 0.6 mg of uniformly 15N-labeled IL-8 to the unlabeled CXCR1 constructs or 1 mg of labeled ND1-38 to the unlabeled IL-8 in a final molar ratio of 1:1. The pH of the IL-8: 1TM1-72 complex was adjusted to 4.7 to increase the sample solubility while the pH of the other samples was 5.5.

NMR spectroscopy

The solution NMR experiments were performed at 40°C on a Bruker DRX 600 MHz spectrometer equipped with 5 mm triple-resonance cryoprobe with z-axis gradient. Heteronuclear solution NMR experiments were performed on uniformly 15N-labeled or uniformly 13C/15N-double-labeled samples with a protein concentration of 0.1 mM. One-dimensional 15N edited 1H NMR spectra resulted from signal averaging of 128 transients. Two-dimensional 1H-15N HSQC spectra were obtained on uniformly and selectively 15N-labeled samples. Triple resonance HNCA and HNCOCA experiments were performed on 13C/15N double-labeled IL-8 and ND1-38 for resonance assignments. The chemical shift perturbations by addition of unlabeled samples were calculated using the equation

equation M1

where ΔδH is the change in the backbone amide proton chemical shift and ΔδN is the change in backbone amide nitrogen chemical shift.

The solid-state 15N NMR spectra were obtained at 40°C on a 700 MHz Bruker Avance spectrometer. The homebuilt 1H/15N double-resonance probe used in the experiments had a 5 mm inner diameter solenoid coil tuned to the 15N frequency and an outer MAGC “low E” coil tuned to the 1H frequency.33 The one-dimensional 15N chemical shift NMR spectra were obtained by spin-lock cross-polarization with a contact time of 1 ms, a recycle delay of 6 s, and an acquisition time of 10 ms. 4096 transients were signal averaged for each spectrum, and an exponential function corresponding to line broadening of 100 Hz was applied to each free induction decay prior to Fourier transformation. The NMR data were processed using the programs NMRPipe/NMRDraw.34 The chemical shift frequencies were externally referenced to 15N-labeled solid ammonium sulfate, defined as 26.8 ppm, which corresponds to the signal from liquid ammonia at 0 ppm.


  • The interaction between IL-8 and CXCR1 was dissected using expressed and isotopically labeled monomeric IL-8 and four different constructs of CXCR1, including the full-length receptor and the N-terminal 39 amino acid residues.
  • Both isotropic (q=0.1) and magnetically aligned (q=3.2) bicelles were used to study the interactions of IL-8 and CXCR1.
  • The use of both solution NMR and OS solid-state NMR enabled the samples to be studied in aqueous solution, isotropic bicelles, and magnetically aligned bilayers, which was highly informative about their binding and dynamics.
  • We were able to demonstrate that the essential species is the ternary complex of IL-8, CXCR1, and the phospholipid bilayer.

Supplementary Material



This research was supported by grants from the National Institutes of Health, and utilized the Biomedical Technology Resource for NMR Molecular Imaging of Proteins at the University of California, San Diego, which is supported by grant P41EB002031. F.C. was supported by postdoctoral fellowships from the Swiss National Science Foundation (PBBSP3-123151) and the Novartis Foundation, formerly the Ciba-Geigy Jubilee Foundation.

Abbreviations used

the first trans membrane helix domain corresponding to the residues 1-72 of CXCR1
G-protein coupled receptor
(4-(2hydroxyethyl)-1-piperazineethanesulfonic acid
modified Alderman-Grant coil
the N-terminal extracellular domain corresponding to the residues 1-38 of CXCR1
nuclear Overhauser effect
N-terminal truncated construct corresponding to the residues 39-350 of CXCR1
OS solid-state NMR
oriented sample solid-state NMR


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