doi:10.1016/j.jmb.2011.08.025 J. Mol. Biol. (2011) 414, 194–203

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Interactions of Interleukin-8 with the HumanChemokine Receptor CXCR1 in PhospholipidBilayers by NMR Spectroscopy

Sang Ho Park, Fabio Casagrande, Leah Cho, Lauren Albrechtand Stanley J. Opella⁎Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive,La Jolla, CA 92093-0307, USA

Received 3 March 2011;received in revised form19 July 2011;accepted 11 August 2011Available online12 October 2011

Edited by A. G. Palmer III

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

*Corresponding author. E-mail addrAbbreviations used: 1TM1–72, the

helix domain corresponding to residDHPC, 1,2-dihexanoyl-sn-glycero-3-pDMPC, 1,2-dimyristoyl-sn-glycero-3-GPCR, G-protein-coupled receptor; IND1–38, the N-terminal extracellular dto residues 1–38 of CXCR1; NT39–350,construct corresponding to residuessolid-state NMR, oriented sample soheteronuclear single quantum cohere

0022-2836/$ - see front matter © 2011 E

CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator ofimmune and inflammatory responses. Strategically located in the cellmembrane, CXCR1 binds to IL-8 with high affinity and subsequentlytransduces a signal across the membrane bilayer to a G-protein-activatedsecond messenger system. Here, we describe NMR studies of theinteractions between IL-8 and human CXCR1 in lipid environments.Functional full-length and truncated constructs of CXCR1 and full-lengthIL-8 were uniformly 15N-labeled by expression in bacteria followed bypurification and refolding. The residues responsible for interactionsbetween IL-8 and the N-terminal domain of CXCR1 were identified byspecific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicellesdisappeared completely when CXCR1 in lipid bilayers was added in a 1:1molar ratio, indicating that binding to the receptor-containing bilayersimmobilizes IL-8 (on the ∼105 Hz timescale) and broadens the signalsbeyond detection. The same solution NMR signals from IL-8 were lessaffected by the addition of N-terminal truncated CXCR1 in lipid bilayers,demonstrating that the N-terminal domain of CXCR1 is mainly responsiblefor binding to IL-8. The interaction is tight enough to immobilize IL-8 alongwith the receptor in phospholipid bilayers and is specific enough to result inwell-aligned samples in oriented sample solid-state NMR spectra. Acombination of solution NMR and solid-state NMR studies of IL-8 in thepresence of various constructs of CXCR1 enables us to propose a model forthe multistep binding process.

© 2011 Elsevier Ltd. All rights reserved.

ess: sopella@ucsd.edu.first transmembraneues 1–72 of CXCR1;hosphocholine;phosphocholine;L-8, interleukin-8;omain correspondingN-terminal truncated39–350 of CXCR1; OSlid-state NMR; HSQC,nce.

lsevier Ltd. All rights reserve

Introduction

The chemokine system regulates many biologicaland pathological processes, including inflammation,embryogenesis, metastasis, host defense againstinfection, and innate immunity.1 Its broad role inregulation is accomplished through the binding ofspecific chemokines to their respective G-protein-coupled receptors (GPCRs). For example, the releaseof the chemokine interleukin-8 (IL-8) by several celltypes is a response to an inflammatory stimulus and

d.

195Interactions of Interleukin-8 with CXCR1

results in the migration of leukocytes, includingneutrophils, monocytes, T- and B-lymphocytes, andbasophils, to these sites. IL-8 has also been shown tostimulate self-renewal of breast cancer stem cells invitro.2 In humans, two high-affinity IL-8 receptors,CXCR1 and CXCR2, have been characterized,3,4 andCXCR1 has been identified as a target for blockingthe formation of breast cancer stem cells that drivetumor growth and metastasis.5

CXCR1 belongs to the family of chemokinereceptors with seven transmembrane helices thatcouple to heterotrimeric G-proteins for signaltransduction.6 We have demonstrated the expres-sion in Escherichia coli, and purification and refold-ing of functional full-length CXCR1, and numerousconstructs of the receptor, including N-terminaltruncated CXCR1 (NT39–350), C-terminal truncatedCXCR1 (CT1–319), both N- and C-terminal double-truncated CXCR1 (DT23–319), the first transmem-brane helix domain of CXCR1 (1TM1–72), and theN-terminal extracellular domain (ND1–38) withoutany residues associated with the first transmem-brane helix.7,8 We have also characterized the localand global dynamics of full-length CXCR1 inmembrane environments using a combination ofsolution NMR and solid-state NMR techniques.9

The mechanisms by which chemokines modulatespecific biological activities are central to under-standing how GPCRs transmit signals through themembrane bilayer to the interior of the cell.Previously, solution NMR spectroscopy has beenused to characterize the structure of IL-8 alone10–12

and bound to synthetic peptides with sequencescorresponding to portions of the N-terminal domainof CXCR1.12,13 Solution NMR is feasible in thesesituations because of the small size and highsolubility of IL-8 and the peptides derived fromthe N-terminal sequence of CXCR1. These studieshave identified a probable location on IL-8 thatinteracts with the N-terminal domain of CXCR1;however, these model systems lack several essentialcomponents of the biological system, namely, theadditional residues present in full-length GPCR andthe planar lipid bilayer environment where thereceptor resides. For example, the earlier studiesusing relatively short synthetic peptides could notdetect interactions with extracellular loops or otherregions of CXCR1 or the effects of lipid bilayers onthe structures, dynamics, and interactions of CXCR1and IL-8.Here, we describe studies that use uniformly 15N-

labeled full-length CXCR1, several of its truncatedconstructs, two versions of its N-terminal domain,and native IL-8 in both free and bound states.Through utilization of both solution NMR and solid-state NMR experiments, it was possible to monitorthe proteins in a wide range of lipid environments,including phospholipid bilayers. Appropriate con-trol experiments on both of the proteins in the

various lipid environments enable us to propose amultistep model for the interactions between IL-8 and CXCR1 in lipid bilayers.

Results

Interaction of the N-terminal domain of CXCR1with 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 oriented sample solid-stateNMR (OS solid-state NMR) spectrum of uniformly15N-labeled ND1–38 in magnetically aligned bilayersdemonstrates that the sample is well aligned on thesurface of the bilayers (Fig. 1a). The signals thatresult from cross-polarization neither are centeredat the isotropic frequency nor have the appearanceof a powder pattern, providing strong evidence forthe existence of specific interactions between thephospholipids and amino acid residues in the N-terminal domain of CXCR1. As a control, IL-8 wassubject to cross-polarization in the presence ofmagnetically aligned bilayers not containing aconstruct of CXCR1, and as expected, no NMRsignals were observed (data not shown) because IL-8 is water soluble and does not interact withphospholipids.The 1H/15N heteronuclear single quantum coher-

ence (HSQC) solution NMR spectrum of ND1–38 inaqueous buffer (Fig. 1b, black contours) has a verylimited dispersion of 1H amide chemical shifts(b1 ppm), which is typical of relatively smallpolypeptides with little or no secondary or tertiarystructure. Moreover, no homonuclear 1H/1H nu-clear Overhauser enhancement cross peaks could beobserved in standard two-dimensional experiments.In contrast, IL-8 yields a well-resolved solutionNMR spectrum that is typical of a native globularprotein, since it has a wide dispersion (N6 ppm) of1H amide chemical shifts and relatively narrow linewidths (Fig. S1b).Compared to aqueous solution, there are signifi-

cant chemical shift changes and broadening of asubset of backbone amide signals of ND1–38 includ-ing the side-chain signal of Trp10 when lipids areadded to the sample (Fig. 1c and Fig. S1a). Incontrast, IL-8 does not interact with lipid bilayers,and therefore, no significant spectral changesincluding to the side-chain signal of Trp57 wereobserved in the presence of phospholipid bilayers(Fig. S1b). This is consistent with the OS solid-stateNMR result on IL-8 alone in the presence of lipidbilayers.The samples made from mixtures of long-chain

phospholipids [e.g., 1,2-dimyristoyl-sn-glycero-3-

Fig. 1. Membrane interaction ofND1–38 and dissociation of the ND1–

38/IL-8 complex from the mem-brane. (a and b) 15N chemicalshift OS solid-state NMR spectra ofuniformly 15N-labeled ND1–38 alone(a) and in complex (b) with unla-beled IL-8 in q=3.2 bicelles. (c and d)1H/15N HSQC solution NMR spec-tra of uniformly 15N-labeled ND1–38alone (c) and in complex (d) withunlabeled IL-8 in aqueous buffer(black contours and one-dimension-al spectrum) and in q=3.2 bicelles(red contours and one dimensional-spectrum). The side-chain signal ofTrp10 residue is indicated. One-dimensional 15N-edited 1H solutionNMR spectra are aligned along thetopof the corresponding two-dimen-sional spectra to compare the signalintensities. The molar ratio of thecomplex was 1:1.

196 Interactions of Interleukin-8 with CXCR1

phosphocholine (DMPC)] and short-chain phospho-lipids [e.g., 1,2-dihexanoyl-sn-glycero-3-phospho-choline (DHPC)] have their molar ratio (long/short) characterized by the parameter “q” and arereferred to as “bicelles.” These protein-containinglipid mixtures enable the structures and dynamics ofthe proteins to be characterized by solution NMRand solid-state NMR experiments; q values less thanabout 1.5 result in isotropic bicelles that aregenerally suitable for solution NMR experiments,and those with values greater than about 2.5 formmagnetically alignable bilayers that immobilize theprotein and require solid-state NMR methods toobtain high-resolution spectra.14–17

In isotropic q=0.1 bicelles, the largest chemicalshift changes were observed primarily near the N-terminus (residues 2–16) of ND1–38 (Fig. S1a). Inmagnetically aligned q=3.2 bilayer samples, themost affected signals, including that from the Trp10side chain, were broadened beyond detection insolution NMR spectra (Fig. 1c, red contours). Thissignificant broadening of the first 16 residues ofCXCR1 does not result from weak alignment of theprotein in the liquid crystalline phase but ratherfrom the interactions with the lipid bilayers, since ina control experiment, all the signals that were onlyslightly broadened could be observed when ND1–38

was weakly aligned using fd bacteriophage particlesin aqueous buffer solution.

Dissociation of the N-terminal domain of CXCR1bound to IL-8 from membranes

The spectra of IL-8 bound to ND1–38 in lipidbilayers provide insights into the ternary complex ofIL-8, CXCR1, and phospholipid bilayers (Fig. 1b andd). There were no significant chemical shift changesin the solution NMR spectrum of the ND1–38 boundto IL-8 when lipid bilayers were added to theaqueous buffer. Remarkably, the signals of freeND1–38 that were broadened out due to themembrane interaction (Fig. 1c, red contours) reap-pear when IL-8 is bound to ND1–38, including theTrp10 side-chain signal (Fig. 1d, red contours).Overall, the line widths of the signals from ND1–38bound to IL-8 are only slightly broader than those offree ND1–38. Taken together, these results demon-strate that ND1–38 does not interact with lipidbilayers when bound to IL-8, and IL-8 does notinteract with bilayers in the absence of the N-terminal domain of CXCR1. The inability, despiteextensive efforts, to obtain solid-state NMR signalsfrom ND1–38 when it is complexed with IL-8 in thepresence of aligned phospholipid bilayers further

197Interactions of Interleukin-8 with CXCR1

supports the finding that the binding of IL-8 resultsin the dissociation of the N-terminal domain ofCXCR1 from phospholipid bilayers (Fig. 1b).

Binding site mapping of the IL-8 andCXCR1 complex

The backbone resonance assignments of free IL-8 under the experimental conditions used here weremade by comparisons to the previously assignedspectra 10 and confirmed by comparisons with1H/15N HSQC spectra of selectively Leu, Ile, Val,and Phe 15N-labeled samples as well as convention-al triple-resonance experiments performed on uni-formly 13C/15N-labeled samples.The amino acid residues that form the binding

sites of IL-8 and of ND1–38 were identified bymapping the chemical shift perturbations resultingfrom complex formation between one uniformly15N-labeled polypeptide in the presence of itsunlabeled counterpart. The expanded region of1H/15N HSQC solution NMR spectra of uniformly15N-labeled IL-8 shows the specific chemical shiftperturbation of backbone amide resonances follow-ing the addition of unlabeled ND1–38 (Fig. 2a). Theplot of chemical shift changes as a function ofresidue number indicates that relatively largechemical shift changes (N0.06 ppm) are observed

Fig. 2. Interaction of IL-8 with truncated CXCR1 constructsspectra: (a) uniformly 15N-labeled IL-8 alone (black contours)aqueous buffer; (b) uniformly 15N-labeled IL-8 alone (blackcontours) in q=0.1 isotropic bicelles; (c) uniformly 15N-labelvarying amounts of unlabeled IL-8 in aqueous buffer. The mocontours), 0.5 (blue contours), and 1 (red contours), respectivelysignals as a function of residue number: (d) plot of IL-8 by ad8 monomer; (e) plot of IL-8 by addition of an equimolar concenas a function of the residue number by addition of 0.25 (green),

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 61and 62 in the C-terminal helix (Fig. 2d). Thisidentifies the regions of IL-8 that interact with theN-terminal domain of CXCR1. These findings aresimilar to those from previous studies performedwith a synthetic peptide corresponding to the first 40residues of the N-terminal domain of CXCR118 andwith a 17-residue peptide, corresponding to residues9–29 of CXCR1 where residues 15–19 were replacedwith a single six-amino hexanoic acid moiety.13

It has been reported that not only the N-terminaldomain but also the extracellular loops of CXCR1are involved in the interaction with IL-8.19 Thespectral changes in IL-8 by the addition ofN-terminal truncated CXCR1 (NT39–350) in q=0.1isotropic bicelles provide evidence for the specificinteractions between IL-8 and extracellular loops ofCXCR1 (Fig. 2b). Although the extent of thechemical shift perturbations of IL-8 by NT39–350was not as large as those by ND1–38, significantline broadening of the signals, except the first sixN-terminal residues, and relatively large chemicalshift changes in Leu17 and Lys23 of IL-8 wereobserved (Fig. 2e).The binding site of the N-terminal region of

CXCR1 has been characterized by the measurement

. (a–c) Expanded region of 1H/15N HSQC solution NMRand in complex with unlabeled ND1–38 (red contours) incontours) and in complex with unlabeled NT39–350 (reded ND1–38 alone (black contours) and in the presence oflar ratios of the IL-8 monomer to ND1–38 were 0.25 (green. (d–f) Chemical shift perturbation plot of backbone amidedition of an equimolar concentration of ND1–38 to the IL-tration of NT39–350 to the IL-8 monomer; (f) plot of ND1–380.5 (blue), and 1 (red) ratios of the IL-8 monomer to ND1–38.

Fig. 3. Interaction of IL-8 with full-length CXCR1. 15N-edited 1H solutionNMRspectra of uniformly 15N-labeled IL-8 in the presence of unlabeled full-length CXCR1 in q=0.1isotropicbicelles.Themolar ratiosofCXCR1to IL-8monomerare listed on the right side of their respective spectra.

198 Interactions of Interleukin-8 with CXCR1

and analysis of intermolecular nuclear Overhauserenhancements observed between IL-8 and the 17-residue peptide derived from CXCR1 describedabove.13 Here, we take advantage of havingprepared an isotopically labeled polypeptide bybacterial expression corresponding to theN-terminaldomain of CXCR1 to map the binding site usingheteronuclear solution NMR experiments. Thechanges in the spectrum of ND1–38 resulting fromthe addition of unlabeled IL-8 have the characteris-tics of “fast exchange” on the timescales of the 1H

Fig. 4. Interaction of IL-8 with three constructs of CXCR1 iNMR spectra of uniformly 15N-labeled IL-8 bound to the conCXCR1; (b) the first transmembrane helix domain of CXCR1 (molar ratio of IL-8 to CXCR1 in each sample was 1:1.

and 15N chemical shifts. With increasing concentra-tions of IL-8, the amide resonances of the affectedresidues shift incrementally from the frequenciesobserved in the free state to those of the fully boundstate (Fig. 2c). The chemical shift frequencies stopchanging when approximately one equivalent ofthe unlabeled IL-8 monomer has been added to thesolution containing labeled ND1–38 (Fig. 2f). Thebinding affinity of ND1–38 and IL-8 was determinedby treating the binding-induced chemical shiftchanges as a titration.20 The Kd is approximately 70μM under these conditions. Previously, N-terminalfragments of CXCR1 have been shown to bind IL-8 with an affinity 3–5 orders of magnitude weakerthan that of the full-length receptor.13,18

Binding of IL-8 to full-length CXCR1 inmembraneenvironments

Interactions of IL-8 with polypeptides whosesequences are derived from the N-terminal regionof CXCR1 have been described previously.13,18,21However, information about the interaction of IL-8 with full-length CXCR1 is scarce largely becauseof the experimental difficulties encountered in thestudy of large membrane proteins in phospholipidbilayers. We have developed protocols for theexpression, purification, and refolding of variousCXCR1 constructs in phospholipid bilayers includ-ing the full-length protein.7,8 This enables us tostudy the interactions of IL-8 with full-length andtruncated constructs of CXCR1 in membraneenvironments.Figure 3 shows the effects of adding increasing the

amounts of CXCR1 in bilayers to a q=0.1 isotropicbicelle solution containing uniformly 15N-labeledIL-8. In the absence of the receptor-containingbilayers, the 15N-edited 1H solution NMR spectrumof the amide region has narrow and well-dispersed

n phospholipid bilayers. 15N chemical shift OS solid-statestructs of CXCR1 in q=3.2 aligned bicelles: (a) full-length1TM1–72); (c) N-terminal truncated CXCR1 (NT39–350). The

199Interactions of Interleukin-8 with CXCR1

resonances, typical of a small globular protein inaqueous solution. As the addition of the receptorapproaches a 1:1 molar ratio of CXCR1:IL-8, nearlyall signals from labeled IL-8 broaden systematicallyand disappear into the baseline, with the exceptionof a few signals that have been assigned to residuesnear the N- and C-termini. The result was moredramatic in lipid bilayers, because with CXCR1 inproteoliposomes at a 1:1 molar ratio with IL-8, all ofthe IL-8 signals disappear as a result of their immo-bilization upon binding to the CXCR1-containingbilayers. Refolded CXCR1 prepared by our methodshas been shown to bind IL-8 with an affinity (Kd of1–5 nM) and to couple to G-protein (EC50 ∼1 nM),7,8

which are similar to the values previously reportedin the literature.3

Critical role of the N-terminal domain of CXCR1for IL-8 binding

Comparisons of 15N chemical shift OS solid-stateNMR spectra of uniformly 15N-labeled IL-8 boundto unlabeled full-length CXCR1 and constructsconsisting of the first transmembrane helix domain(1TM1–72) and the N-terminal truncated (NT39–350)receptors in lipid bilayers are shown in Fig. 4. Theseresults demonstrate that the N-terminal domain ofCXCR1 is mainly responsible for the binding of IL-8.The OS solid-state NMR signals of IL-8 were intenseand well resolved when IL-8 was added to full-length and 1TM1–72 receptors aligned in lipidbilayers, demonstrating that their interaction isstrong enough to immobilize and align IL-8 alongwith the receptor at a unique orientation in themagnetically aligned bilayers (Fig. 4a and b). As acontrol, no IL-8 signals could be observed in OSsolid-state NMR experiments in a sample containinglabeled IL-8 and an unlabeled NT39–350 (Fig. 4c).Since binding to the receptor is necessary toimmobilize and align the IL-8, this suggests thatthe binding site is predominantly located in theN-terminal region of the receptor.

Discussion

Comparisons between the solution NMR andsolid-state NMR spectra of ND1–38 alone andbound to IL-8 provide information about theinfluence of the lipid bilayer on interactions of theN-terminal domain of CXCR1 and IL-8. The N-terminal region of CXCR1 determines the specificityand affinity for IL-8.22,23 Recently, a 34-residuepeptide with a sequence corresponding to the N-terminal residues of rabbit CXCR1 was shown tointeract with the membrane surface by monitoringfluorescence of two tryptophan residues of thepeptide.24 Both human and rabbit CXCR1 receptorshave similar affinity and specificity for human IL-

8,21 and their N-terminal domains have highsequence homology (Fig. S2). Tryptophan residuesare commonly found near the membrane surface,since the polar amide group and hydrophobic ringstructure of this amino acid facilitate its localizationat the polar/apolar interface.25 Significantly, signalsfrom both the backbone and the side chain of thetryptophan residue in ND1–38 are broadened beyonddetection in the presence of lipid bilayers (Fig. 1c),suggesting that the tryptophan residue may serve asan anchor on the membrane surface. The tryptophanresidues located in the N-terminal domain of rabbitCXCR1, one of which is located in the same positionas a tryptophan in the human CXCR1 sequence,have been shown to be involved directly inmembrane interactions.24

The chemical shift perturbation plot for labeled IL-8 in Fig. 2d obtained by the addition of unlabeledND1–38 shows substantial changes in three regionsof the primary sequence. The residues that contrib-ute to the binding cleft identified in the three-dimensional structure of IL-8 were the ones moststrongly affected by the interaction with ND1–38. Thecentral region of the ND1–38 primary sequence(residues 18–27) was most strongly affected bybinding to IL-8. This suggests that ND1–38 mayadopt an extended conformation when complexedto IL-8. Although the proline residues of ND1–38were not monitored in our experiments, alanine-scanning studies have shown that the two prolines,21 and 29, as well as Tyr27 contribute to theinteractions with IL-8, suggesting that the hydro-phobic characteristics of these residues play roles inbinding to the N-terminal domain of CXCR1.26

Many studies of chemokines and their interactionswith receptors have concluded that one or more ofthe extracellular loops of the receptors are involved.In particular, alanine-scanning experiments haveshown that the third and fourth extracellular loopsof CXCR1 are involved in the binding to IL-8.19 Anoverall broadening of solution NMR signals of IL-8 in the presence of 1TM1–72 (data not shown) andNT39–350 (Fig. 2b) at a molar ratio of 1:1 wasobserved, but in both cases, the signals were lessaffected than those of IL-8 in the presence of the full-length receptor (Fig. 3). Two possible reasons for thisdifference are that the binding of IL-8 to 1TM1–72 isnot as tight as for the full-length receptor or that thebinding is as tight as full-length receptor, but thesmaller size of the IL-8 and 1TM1–72 complex(∼18 kDa) reorients faster than IL-8 and the full-length complex (∼52 kDa) in isotropic q=0.1bicelles. In the case of the N-terminal truncatedreceptor, the molecular mass of NT39–350 is reducedby only 10% compared to the full-length receptor;thus, the reduction in rotational correlation time isunlikely to be sufficient to account for the spectralchanges. It may be that the changes are a manifes-tation of weak interactions of IL-8 to extracellular

200 Interactions of Interleukin-8 with CXCR1

loop regions of the receptor without the contribu-tions from the missing residues in the N-terminaldomain of the receptor.The role of dimerization of IL-8 in binding CXCR1

is not fully understood, but recent studies haveshown that the IL-8 monomer binds to the N-terminal domain of CXCR1 with higher affinity thanthe IL-8 dimer.27,28 We used only the monomericform of CXCR1, and in all of our experiments, thespectral changes stopped when an approximatelyequimolar concentration of CXCR1 monomer to theIL-8 monomer was achieved. These results suggestthat one molecule of CXCR1 binds to one moleculeof the IL-8 monomer. Since IL-8 exists as a stablehomodimer in an aqueous solution, it is possiblethat the chemical shift perturbation of IL-8 uponbinding to CXCR1 constructs results not only fromthe direct interaction between them but also fromthe dimer-to-monomer transition of IL-8.It is essential to obtain atomic-resolution structural

details about how IL-8 interacts with its high-affinitymembrane-embedded receptors in order to under-stand the first step of the complex signaling cascade.In the meantime, we interpret the NMR resultsdiscussed above in terms of a multistep series ofinteractions between IL-8 and CXCR1 with signifi-cant contributions from the phospholipid bilayers(Fig. 5). Thus,we propose that the ternary complex ofIL-8/CXCR1/bilayer is an essential species.In the first step, the N-terminal domain of

CXCR1, which has many characteristics of a

Fig. 5. Model of IL-8 interacting with CXCR1 in membranflexible yet structured by interacting with the surface of the mof the domain is mainly involved in membrane interaction, anmembrane. Step 2: The strong interaction between the N-termembrane surface. Step 3: The N-terminal domain in compleextracellular loops, potentially creating a conformational chmonomer from the IL-8 dimer structure (Protein Data Bank ID49, 50, 61, and 62) whose chemical shifts were perturbed sigCXCR1 are shown as red spheres.

peripheral membrane protein, interacts transientlywith the membrane surface and adopts a rela-tively well-defined yet still flexible structure thatmay contribute to receptor selectivity. Our NMRdata on the N-terminal domain of CXCR1 in theabsence and presence of phospholipid bicellesclearly demonstrate the significant effects of themembrane environment on the structure anddynamics of this domain (Fig. 1). In particular,the Trp10 side chain is likely to be embedded inthe bilayer.In the second step, after binding to IL-8, the N-

terminal domain dissociates from the membranesurface. Upon interaction with IL-8, the solutionNMR signals of the N-terminal domain that werecompletely broadened out due to the membraneinteraction (step 1) reappeared as a result ofdissociation of the domain from the membrane(Fig. 1d). The complementary OS solid-state NMRspectrum of the domain in complex did not yieldany signals, which also demonstrates that thecomplex is no longer immobilized by interactionswith the membrane (Fig. 1b).In the third step, the complex of IL-8 and the N-

terminal domain rearranges to engage a secondbinding site on the receptor, most likely involvingone or more extracellular loops (Fig. 2b and e). Thisstep might be the trigger for the conformationalchanges in the receptor needed to activate secondarysignaling cascades. This does not exclude thepossibility that IL-8 interacts simultaneously with

es. Step 1: The N-terminal domain of CXCR1 (green) isembrane, contributing to receptor selectivity. The first halfd Trp10 serves as an anchor on the extracellular side of theminal domain and IL-8 dissociates the domain from thex with IL-8 is translated to the second binding site of theange in CXCR1 for subsequent G-protein activation. A2IL8) is represented. The residues of IL-8 (12, 17, 20, 44, 48,nificantly by interaction with the N-terminal domain of

201Interactions of Interleukin-8 with CXCR1

the N-terminal domain and extracellular loops of thereceptor.A two-site mechanism of chemokine receptor

interaction in which the N-terminal domain andextracellular loop in the receptor are involved in theligand interaction has been proposed based on thevarious structure–function studies reviewed byRajagopalan and Rajarathnam.29 Although it is notfully understood how the two-site mechanismmediates affinity, selectivity, and activation of thereceptor, the N-terminal residues of the receptor areshown to be essential for both binding affinity andreceptor selectivity.22 The OS solid-state NMR datapresented here show that the N-terminal domain ofCXCR1 is mainly responsible for the strong interac-tion with IL-8 (Fig. 4).It has beenproposed that the chemokineN-terminal

“ELR” motif interacts with the extracellular loops ofthe receptor.30,31 Recently, the highly dynamicN-terminus including the ELRmotif of the chemokineSDF-1 has been proposed to play a crucial role in theinteraction with its receptor CXCR4.32 However, wedo not observe experimental NMR evidence that theN-terminal ELRmotif of IL-8 interactswith full-lengthor N-terminal truncated CXCR1. This may be due todifferences between the two receptors, or it mayrequire future studies of the structures and mecha-nisms of GPCRs to fully sort out.The interactions between ligands and their mem-

brane-embedded receptors, especially GPCRs, arethe first step in initiating the complex cascades ofprotein interactions known to regulate physiologicalprocesses in mammals. Here, we demonstrate thatthe interaction between IL-8 and its receptor, CXCR1,must be analyzed in the context of the phospholipidbilayer environment. Solid-state NMR spectroscopyis unique in providing atomic-resolution informationabout membrane proteins and their complexes inphospholipid bilayers under conditionswhere signaltransduction occurs. The resulting NMR data enableus to propose a model for the interactions betweenIL-8 and CXCR1 that involve the phospholipidbilayer, IL-8, the N-terminal domain of CXCR1,and residues in inter-helical loops near theC-terminus.In summary, we conclude that the membrane bilayerplays a role that is as important as the structuralfeatures of the two protein components in theinteractions of IL-8 and CXCR1 in the first step oftransducing biological signals.

†www.avantilipids.com

Materials and Methods

Sample preparation

IL-8 was expressed and purified as describedpreviously.22 Full-length CXCR1 and three truncatedconstructs including N-terminal truncated CXCR1

(NT39–350), the first transmembrane helix domain ofCXCR1 (1TM1–72), and the N-terminal extracellular do-main of CXCR1 (ND1–38) were expressed, purified, andrefolded as described previously.7,8 The amino acidsequences of the CXCR1 constructs are shown in Support-ing Information. The amino acid sequence of ND1–38substitutes Ser for Cys at position 30 to prevent compli-cations due to intermolecular disulfide bond formation.For the solution NMR experiments, the concentration of

IL-8 and ND1–38 polypeptides was 0.1 mM, in 20 mMHepes, at pH 5.5, in 400 μl of 90% H2O/10% 2H2O. Theprotein-containing bicelle samples of IL-8 and ND1–38were prepared by dissolving the lyophilized polypeptidesdirectly into premixed solutions containing DMPC andDHPC phospholipids. The lipids were obtained fromAvanti Polar Lipids†. The isotropic (q=0.1) and magnet-ically alignable (q=3.2) samples contain 10% DHPC (w/v)and 10% DMPC (w/v), respectively. The samples of theCXCR1 constructs, except for the soluble ND1–38 poly-peptide, were prepared from proteoliposome pellets [20%(w/v) lipid] in which 1 mg of the polypeptide wasreconstituted into a solution containing 10 mg of DMPC.For the titration experiments, a stock solution of theunlabeled proteins under the same buffer conditions wasadded to the uniformly 15N-labeled proteins so that thefinal 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 mixturecontaining 20% DMPC (w/v) and 20 mM Hepes, atpH 5.5. The complex was formed by adding 0.6 mg ofuniformly 15N-labeled IL-8 to the unlabeled CXCR1constructs 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: 1TM1–72complex was adjusted to 4.7 to increase the samplesolubility, while the pH of the other samples was 5.5.

NMR spectroscopy

The solution NMR experiments were performed at40 °C on a Bruker DRX 600-MHz spectrometer equippedwith 5-mm triple-resonance cryoprobe with z-axisgradient. Heteronuclear solution NMR experiments wereperformed on uniformly 15N-labeled or uniformly13C/15N-double-labeled samples with a protein concen-tration of 0.1 mM. One-dimensional 15N-edited 1H NMRspectra resulted from signal averaging of 128 transients.Two-dimensional 1H/15N HSQC spectra were obtainedon uniformly and selectively 15N-labeled samples. Triple-resonance HNCA and HNCOCA experiments wereperformed on 13C/15N-double-labeled IL-8 and ND1–38for resonance assignments. The chemical shift perturba-tions by addition of unlabeled samples were calculatedusing the equation

Dy =

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiDyΗð Þ2 + DyN =5ð Þ2

2

s

where ΔδH is the change in the backbone amide protonchemical shift and ΔδN is the change in backbone amidenitrogen chemical shift.

202 Interactions of Interleukin-8 with CXCR1

The solid-state 15N NMR spectra were obtained at 40 °Con a 700-MHz Bruker Avance spectrometer. Thehomebuilt 1H/15N double-resonance probe used in theexperiments had a 5-mm-inner-diameter solenoid coiltuned to the 15N frequency and an outer MAGC (modifiedAlderman–Grant coil) “low E” coil tuned to the 1Hfrequency.33 The one-dimensional 15N chemical shiftNMR spectra were obtained by spin-lock cross-polariza-tion with a contact time of 1 ms, a recycle delay of 6 s, andan acquisition time of 10 ms. Transients (4096) were signalaveraged for each spectrum, and an exponential functioncorresponding to line broadening of 100 Hzwas applied toeach free induction decay prior to Fourier transformation.The NMR data were processed using the programsNMRPipe/NMRDraw.34 The chemical shift frequencieswere externally referenced to 15N-labeled solid ammoni-um sulfate, defined as 26.8 ppm, which corresponds to thesignal from liquid ammonia at 0 ppm.

Acknowledgements

This research was supported by grants from theNational Institutes of Health and utilized the Biotech-nology Resource Center for NMRMolecular Imagingof Proteins at the University of California, San Diego,which is supported by grant P41EB002031. F.C. wassupported by postdoctoral fellowships from the SwissNational Science Foundation (PBBSP3-123151) andthe Novartis Foundation, formerly the Ciba-GeigyJubilee Foundation.

Supplementary Data

Supplementary data to this article can be foundonline at doi:10.1016/j.jmb.2011.08.025

References

1. Fernandez, E. J. & Lolis, E. (2002). Structure, function,and inhibition of chemokines. Annu. Rev. Pharmacol.Toxicol. 42, 469–499.

2. Visvader, J. E. & Lindeman, G. J. (2008). Cancer stemcells in solid tumours: accumulating evidence andunresolved questions. Nat. Rev., Cancer, 8, 755–768.

3. Holmes, W. E., Lee, J., Kuang, W. J., Rice, G. C. &Wood, W. I. (1991). Structure and functional expres-sion of a human interleukin-8 receptor. Science, 253,1278–1280.

4. Murphy, P. M. & Tiffany, H. L. (1991). Cloning ofcomplementary DNA encoding a functional humaninterleukin-8 receptor. Science, 253, 1280–1283.

5. Ginestier, C., Liu, S., Diebel, M. E., Korkaya, H., Luo,M., Brown, M. et al. (2010). CXCR1 blockade selec-tively targets human breast cancer stem cells in vitroand in xenografts. J. Clin. Invest. 120, 485–497.

6. Wu, D., LaRosa, G. J. & Simon, M. I. (1993). G protein-coupled signal transduction pathways for interleukin-8. Science, 261, 101–103.

7. Park, S. H., Prytulla, S., De Angelis, A. A., Brown,J. M., Kiefer, H. & Opella, S. J. (2006). High-resolutionNMR spectroscopy of a GPCR in aligned bicelles.J. Am. Chem. Soc. 128, 7402–7403.

8. Casagrande, F., Maier, K., Kiefer, H., Opella, S. J. &Park, S. H. (2011). Expression and purification of G-protein coupled receptors for NMR structural studies.In Production of Membrane Proteins (Robinson, A. S.,ed.), Wiley-vch, Weinheim, Germany.

9. Park, S. H., Casagrande, F., Das, B. B., Albrecht, L.,Chu, M. & Opella, S. J. (2011). Local and globaldynamics of the G protein-coupled receptor CXCR1.Biochemistry, 50, 2371–2380.

10. Clore, G. M., Appella, E., Yamada, M., Matsushima,K. & Gronenborn, A. M. (1989). Determination of thesecondary structure of interleukin-8 by nuclearmagnetic resonance spectroscopy. J. Biol. Chem. 264,18907–18911.

11. Clore, G. M. & Gronenborn, A. M. (1995). Three-dimensional structures of alpha and beta chemokines.FASEB J. 9, 57–62.

12. Rajarathnam, K., Clark-Lewis, I. & Sykes, B. D. (1995).1H NMR solution structure of an active monomericinterleukin-8. Biochemistry, 34, 12983–12990.

13. Skelton, N. J., Quan, C., Reilly, D. & Lowman, H.(1999). Structure of a CXC chemokine-receptor frag-ment in complex with interleukin-8. Structure, 7,157–168.

14. De Angelis, A. A., Nevzorov, A. A., Park, S. H.,Howell, S. C., Mrse, A. A. & Opella, S. J. (2004). High-resolution NMR spectroscopy of membrane proteinsin aligned bicelles. J. Am. Chem. Soc. 126, 15340–15341.

15. Park, S. H., De Angelis, A. A., Nevzorov, A. A., Wu, C.H. & Opella, S. J. (2006). Three-dimensional structureof the transmembrane domain of Vpu from HIV-1 inaligned phospholipid bicelles. Biophys. J. 91,3032–3042.

16. De Angelis, A. A., Howell, S. C., Nevzorov, A. A. &Opella, S. J. (2006). Structure determination of amembrane protein with two trans-membrane helicesin aligned phospholipid bicelles by solid-state NMRspectroscopy. J. Am. Chem. Soc. 128, 12256–12267.

17. Park, S. H., Marassi, F. M., Black, D. & Opella, S. J.(2010). Structure and dynamics of the membrane-bound form of Pf1 coat protein: implications ofstructural rearrangement for virus assembly. Biophys.J. 99, 1465–1474.

18. Clubb, R. T., Omichinski, J. G., Clore, G. M. &Gronenborn, A. M. (1994). Mapping the bindingsurface of interleukin-8 complexed with an N-termi-nal fragment of the type 1 human interleukin-8 receptor. FEBS Lett. 338, 93–97.

19. Leong, S. R., Kabakoff, R. C. & Hebert, C. A. (1994).Complete mutagenesis of the extracellular domain ofinterleukin-8 (IL-8) type A receptor identifies chargedresidues mediating IL-8 binding and signal transduc-tion. J. Biol. Chem. 269, 19343–19348.

20. Lian, L. & Roberts, G. C. K. (1993). Effects of chemicalexchange on NMR spectra. In NMR of Macromolecules.A Practical Approach (Roberts, G. C. K., ed.),pp. 153–182, Oxford University Press, Oxford, UK.

21. Gayle, R. B., III, Sleath, P. R., Srinivason, S., Birks,C. W., Weerawarna, K. S., Cerretti, D. P. et al. (1993).Importance of the amino terminus of the interleukin-

203Interactions of Interleukin-8 with CXCR1

8 receptor in ligand interactions. J. Biol. Chem. 268,7283–7289.

22. Rajagopalan, L. & Rajarathnam, K. (2004). Ligandselectivity and affinity of chemokine receptor CXCR1.Role of N-terminal domain. J. Biol. Chem. 279,30000–30008.

23. Prado, G. N., Suetomi, K., Shumate, D., Maxwell, C.,Ravindran, A., Rajarathnam, K. & Navarro, J. (2007).Chemokine signaling specificity: essential role for theN-terminal domain of chemokine receptors. Biochem-istry, 46, 8961–8968.

24. Haldar, S., Raghuraman, H., Namani, T., Rajarath-nam, K. & Chattopadhyay, A. (2010). Membraneinteraction of the N-terminal domain of chemokinereceptor CXCR1. Biochim. Biophys. Acta, 1798,1056–1061.

25. Schiffer, M., Chang, C. H. & Stevens, F. J. (1992). Thefunctions of tryptophan residues in membrane pro-teins. Protein Eng. 5, 213–214.

26. Attwood, M. R., Borkakoti, N., Bottomley, G. A.,Conway, E. A., Cowan, I., Fallowfield, A. G. et al.(1996). Identification and characterisation of aninhibitor of interleukin-8: a receptor based approach.Bioorg. Med. Chem. Lett. 6, 1869–1874.

27. Fernando, H., Chin, C., Rosgen, J. & Rajarathnam, K.(2004). Dimer dissociation is essential for interleukin-8 (IL-8) binding to CXCR1 receptor. J. Biol. Chem. 279,36175–36178.

28. Ravindran, A., Joseph, P. R. & Rajarathnam, K. (2009).Structural basis for differential binding of the inter-leukin-8 monomer and dimer to the CXCR1 N-

domain: role of coupled interactions and dynamics.Biochemistry, 48, 8795–8805.

29. Rajagopalan, L. & Rajarathnam, K. (2006). Structuralbasis of chemokine receptor function—a model forbinding affinity and ligand selectivity. Biosci. Rep. 26,325–339.

30. Clark-Lewis, I., Schumacher, C., Baggiolini, M. &Moser, B. (1991). Structure–activity relationships ofinterleukin-8 determined using chemically synthe-sized analogs. Critical role of NH2-terminal residuesand evidence for uncoupling of neutrophil chemotax-is, exocytosis, and receptor binding activities. J. Biol.Chem. 266, 23128–23134.

31. Moser, B., Dewald, B., Barella, L., Schumacher, C.,Baggiolini, M. & Clark-Lewis, I. (1993). Interleukin-8 antagonists generated by N-terminal modification.J. Biol. Chem. 268, 7125–7128.

32. Kofuku, Y., Yoshiura, C., Ueda, T., Terasawa, H.,Hirai, T., Tominaga, S. et al. (2009). Structural basis ofthe interaction between chemokine stromal cell-derived factor-1/CXCL12 and its G-protein-coupledreceptor CXCR4. J. Biol. Chem. 284, 35240–35250.

33. Grant, C. V., Yang, Y., Glibowicka, M., Wu, C. H.,Park, S. H., Deber, C. M. & Opella, S. J. (2009). AModified Alderman–Grant Coil makes possible anefficient cross-coil probe for high field solid-state NMRof lossy biological samples. J. Magn. Reson. 201, 87–92.

34. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G.,Pfeifer, J. & Bax, A. (1995). NMRPipe: a multidimen-sional spectral processing system based on UNIXpipes. J. Biomol. NMR, 6, 277–293.