Opella et al


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Opella et al

  1. 1. doi:10.1016/j.jmb.2011.08.025 J. Mol. Biol. (2011) 414, 194–203 Contents lists available at www.sciencedirect.com Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m bInteractions of Interleukin-8 with the HumanChemokine Receptor CXCR1 in PhospholipidBilayers by NMR SpectroscopySang 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, USAReceived 3 March 2011; CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator ofreceived in revised form immune and inflammatory responses. Strategically located in the cell19 July 2011; membrane, CXCR1 binds to IL-8 with high affinity and subsequentlyaccepted 11 August 2011 transduces a signal across the membrane bilayer to a G-protein-activatedAvailable online second messenger system. Here, we describe NMR studies of the12 October 2011 interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-lengthEdited by A. G. Palmer III IL-8 were uniformly 15N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactionsKeywords: between IL-8 and the N-terminal domain of CXCR1 were identified bymembrane protein; specific chemical shift perturbations of assigned resonances on both IL-solid-state NMR; 8 and CXCR1. Solution NMR signals from IL-8 in q = 0.1 isotropic bicelleslipid bilayer; disappeared completely when CXCR1 in lipid bilayers was added in a 1:1CXCR1; molar ratio, indicating that binding to the receptor-containing bilayersIL-8 immobilizes IL-8 (on the ∼ 10 5 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 enables us to propose a model for the multistep binding process. © 2011 Elsevier Ltd. All rights reserved. *Corresponding author. E-mail address: sopella@ucsd.edu. Introduction Abbreviations used: 1TM1–72, the first transmembranehelix domain corresponding to residues 1–72 of CXCR1; The chemokine system regulates many biologicalDHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; and pathological processes, including inflammation,DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; embryogenesis, metastasis, host defense againstGPCR, G-protein-coupled receptor; IL-8, interleukin-8; infection, and innate immunity. 1 Its broad role inND1–38, the N-terminal extracellular domain corresponding regulation is accomplished through the binding ofto residues 1–38 of CXCR1; NT39–350, N-terminal truncated specific chemokines to their respective G-protein-construct corresponding to residues 39–350 of CXCR1; OS coupled receptors (GPCRs). For example, the releasesolid-state NMR, oriented sample solid-state NMR; HSQC, of the chemokine interleukin-8 (IL-8) by several cellheteronuclear single quantum coherence. types is a response to an inflammatory stimulus and0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.
  2. 2. Interactions of Interleukin-8 with CXCR1 195results in the migration of leukocytes, including various lipid environments enable us to propose aneutrophils, monocytes, T- and B-lymphocytes, and multistep model for the interactions between IL-basophils, to these sites. IL-8 has also been shown to 8 and CXCR1 in lipid bilayers.stimulate 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 and ResultsCXCR1 has been identified as a target for blockingthe formation of breast cancer stem cells that drivetumor growth and metastasis. 5 Interaction of the N-terminal domain of CXCR1 CXCR1 belongs to the family of chemokine with membranesreceptors with seven transmembrane helices thatcouple to heterotrimeric G-proteins for signal We expressed, purified, and characterized the N-transduction. 6 We have demonstrated the expres- terminal extracellular domain of CXCR1 (ND1–38)sion in Escherichia coli, and purification and refold- that corresponds to the first 38 residues of CXCR1.ing of functional full-length CXCR1, and numerous The 15N chemical shift oriented sample solid-stateconstructs of the receptor, including N-terminal NMR (OS solid-state NMR) spectrum of uniformly 15truncated CXCR1 (NT39–350), C-terminal truncated N-labeled ND1–38 in magnetically aligned bilayersCXCR1 (CT1–319), both N- and C-terminal double- demonstrates that the sample is well aligned on thetruncated CXCR1 (DT23–319), the first transmem- surface of the bilayers (Fig. 1a). The signals thatbrane helix domain of CXCR1 (1TM1–72), and the result from cross-polarization neither are centeredN-terminal extracellular domain (ND1–38) without at the isotropic frequency nor have the appearanceany residues associated with the first transmem- of a powder pattern, providing strong evidence forbrane helix. 7,8 We have also characterized the local the existence of specific interactions between theand global dynamics of full-length CXCR1 in phospholipids and amino acid residues in the N-membrane environments using a combination of terminal domain of CXCR1. As a control, IL-8 wassolution NMR and solid-state NMR techniques. 9 subject to cross-polarization in the presence of The mechanisms by which chemokines modulate magnetically aligned bilayers not containing aspecific biological activities are central to under- construct of CXCR1, and as expected, no NMRstanding how GPCRs transmit signals through the signals were observed (data not shown) because IL-membrane bilayer to the interior of the cell. 8 is water soluble and does not interact withPreviously, solution NMR spectroscopy has been phospholipids.used to characterize the structure of IL-8 alone 10–12 The 1H/ 15N heteronuclear single quantum coher-and bound to synthetic peptides with sequences ence (HSQC) solution NMR spectrum of ND1–38 incorresponding to portions of the N-terminal domain aqueous buffer (Fig. 1b, black contours) has a veryof CXCR1. 12,13 Solution NMR is feasible in these limited dispersion of 1H amide chemical shiftssituations because of the small size and high (b 1 ppm), which is typical of relatively smallsolubility of IL-8 and the peptides derived from polypeptides with little or no secondary or tertiarythe N-terminal sequence of CXCR1. These studies structure. Moreover, no homonuclear 1H/ 1H nu-have identified a probable location on IL-8 that clear Overhauser enhancement cross peaks could beinteracts with the N-terminal domain of CXCR1; observed in standard two-dimensional experiments.however, these model systems lack several essential In contrast, IL-8 yields a well-resolved solutioncomponents of the biological system, namely, the NMR spectrum that is typical of a native globularadditional residues present in full-length GPCR and protein, since it has a wide dispersion (N 6 ppm) of 1the planar lipid bilayer environment where the H amide chemical shifts and relatively narrow linereceptor resides. For example, the earlier studies widths (Fig. S1b).using relatively short synthetic peptides could not Compared to aqueous solution, there are signifi-detect interactions with extracellular loops or other cant chemical shift changes and broadening of aregions of CXCR1 or the effects of lipid bilayers on subset of backbone amide signals of ND1–38 includ-the structures, dynamics, and interactions of CXCR1 ing the side-chain signal of Trp10 when lipids areand IL-8. added to the sample (Fig. 1c and Fig. S1a). In Here, we describe studies that use uniformly 15N- contrast, IL-8 does not interact with lipid bilayers,labeled full-length CXCR1, several of its truncated and therefore, no significant spectral changesconstructs, two versions of its N-terminal domain, including to the side-chain signal of Trp57 wereand native IL-8 in both free and bound states. observed in the presence of phospholipid bilayersThrough utilization of both solution NMR and solid- (Fig. S1b). This is consistent with the OS solid-statestate NMR experiments, it was possible to monitor NMR result on IL-8 alone in the presence of lipidthe proteins in a wide range of lipid environments, bilayers.including phospholipid bilayers. Appropriate con- The samples made from mixtures of long-chaintrol experiments on both of the proteins in the phospholipids [e.g., 1,2-dimyristoyl-sn-glycero-3-
  3. 3. 196 Interactions of Interleukin-8 with CXCR1 Fig. 1. Membrane interaction of ND1–38 and dissociation of the ND1– 38/IL-8 complex from the mem- brane. (a and b) 15 N chemical shift OS solid-state NMR spectra of uniformly 15N-labeled ND1–38 alone (a) and in complex (b) with unla- beled IL-8 in q = 3.2 bicelles. (c and d) 1 H/ 15N HSQC solution NMR spec- tra of uniformly 15N-labeled ND1–38 alone (c) and in complex (d) with unlabeled 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 of Trp10 residue is indicated. One- dimensional 15N-edited 1H solution NMR spectra are aligned along the top of the corresponding two-dimen- sional spectra to compare the signal intensities. The molar ratio of the complex was 1:1.phosphocholine (DMPC)] and short-chain phospho- was weakly aligned using fd bacteriophage particleslipids [e.g., 1,2-dihexanoyl-sn-glycero-3-phospho- in aqueous buffer solution.choline (DHPC)] have their molar ratio (long/short) characterized by the parameter “q” and are Dissociation of the N-terminal domain of CXCR1referred to as “bicelles.” These protein-containing bound to IL-8 from membraneslipid mixtures enable the structures and dynamics ofthe proteins to be characterized by solution NMR The spectra of IL-8 bound to ND1–38 in lipidand solid-state NMR experiments; q values less than bilayers provide insights into the ternary complex ofabout 1.5 result in isotropic bicelles that are IL-8, CXCR1, and phospholipid bilayers (Fig. 1b andgenerally suitable for solution NMR experiments, d). There were no significant chemical shift changesand those with values greater than about 2.5 form in the solution NMR spectrum of the ND1–38 boundmagnetically alignable bilayers that immobilize the to IL-8 when lipid bilayers were added to theprotein and require solid-state NMR methods to aqueous buffer. Remarkably, the signals of freeobtain high-resolution spectra. 14–17 ND 1–38 that were broadened out due to the In isotropic q = 0.1 bicelles, the largest chemical membrane interaction (Fig. 1c, red contours) reap-shift changes were observed primarily near the N- pear when IL-8 is bound to ND1–38, including theterminus (residues 2–16) of ND1–38 (Fig. S1a). In Trp10 side-chain signal (Fig. 1d, red contours).magnetically aligned q = 3.2 bilayer samples, the Overall, the line widths of the signals from ND1–38most affected signals, including that from the Trp10 bound to IL-8 are only slightly broader than those ofside chain, were broadened beyond detection in free ND1–38. Taken together, these results demon-solution NMR spectra (Fig. 1c, red contours). This strate that ND1–38 does not interact with lipidsignificant broadening of the first 16 residues of bilayers when bound to IL-8, and IL-8 does notCXCR1 does not result from weak alignment of the interact with bilayers in the absence of the N-protein in the liquid crystalline phase but rather terminal domain of CXCR1. The inability, despitefrom the interactions with the lipid bilayers, since in extensive efforts, to obtain solid-state NMR signalsa control experiment, all the signals that were only from ND1–38 when it is complexed with IL-8 in theslightly broadened could be observed when ND1–38 presence of aligned phospholipid bilayers further
  4. 4. Interactions of Interleukin-8 with CXCR1 197supports the finding that the binding of IL-8 results in three distinct regions of the IL-8 sequence:in the dissociation of the N-terminal domain of residues 12, 17, and 20 in the N-loop; residues 44,CXCR1 from phospholipid bilayers (Fig. 1b). 48, 49, and 50 in the third β-strand; and residues 61 and 62 in the C-terminal helix (Fig. 2d). ThisBinding site mapping of the IL-8 and identifies the regions of IL-8 that interact with theCXCR1 complex N-terminal domain of CXCR1. These findings are similar to those from previous studies performed The backbone resonance assignments of free IL- with a synthetic peptide corresponding to the first 408 under the experimental conditions used here were residues of the N-terminal domain of CXCR1 18 andmade by comparisons to the previously assigned with a 17-residue peptide, corresponding to residuesspectra 10 and confirmed by comparisons with 9–29 of CXCR1 where residues 15–19 were replaced1 H/ 15N HSQC spectra of selectively Leu, Ile, Val, with a single six-amino hexanoic acid moiety. 13and Phe 15N-labeled samples as well as convention- It has been reported that not only the N-terminalal triple-resonance experiments performed on uni- domain but also the extracellular loops of CXCR1formly 13C/ 15N-labeled samples. are involved in the interaction with IL-8. 19 The The amino acid residues that form the binding spectral changes in IL-8 by the addition ofsites of IL-8 and of ND1–38 were identified by N-terminal truncated CXCR1 (NT39–350) in q = 0.1mapping the chemical shift perturbations resulting isotropic bicelles provide evidence for the specificfrom complex formation between one uniformly interactions between IL-8 and extracellular loops of15 N-labeled polypeptide in the presence of its CXCR1 (Fig. 2b). Although the extent of theunlabeled counterpart. The expanded region of chemical shift perturbations of IL-8 by NT39–3501 H/ 15N HSQC solution NMR spectra of uniformly was not as large as those by ND1–38, significant15 N-labeled IL-8 shows the specific chemical shift line broadening of the signals, except the first sixperturbation of backbone amide resonances follow- N-terminal residues, and relatively large chemicaling the addition of unlabeled ND1–38 (Fig. 2a). The shift changes in Leu17 and Lys23 of IL-8 wereplot of chemical shift changes as a function of observed (Fig. 2e).residue number indicates that relatively large The binding site of the N-terminal region ofchemical shift changes (N0.06 ppm) are observed CXCR1 has been characterized by the measurement Fig. 2. Interaction of IL-8 with truncated CXCR1 constructs. (a–c) Expanded region of 1H/ 15N HSQC solution NMRspectra: (a) uniformly 15N-labeled IL-8 alone (black contours) and in complex with unlabeled ND1–38 (red contours) inaqueous buffer; (b) uniformly 15N-labeled IL-8 alone (black contours) and in complex with unlabeled NT39–350 (redcontours) in q = 0.1 isotropic bicelles; (c) uniformly 15N-labeled ND1–38 alone (black contours) and in the presence ofvarying amounts of unlabeled IL-8 in aqueous buffer. The molar ratios of the IL-8 monomer to ND1–38 were 0.25 (greencontours), 0.5 (blue contours), and 1 (red contours), respectively. (d–f) Chemical shift perturbation plot of backbone amidesignals as a function of residue number: (d) plot of IL-8 by addition of an equimolar concentration of ND1–38 to the IL-8 monomer; (e) plot of IL-8 by addition of an equimolar concentration of NT39–350 to the IL-8 monomer; (f) plot of ND1–38as a function of the residue number by addition of 0.25 (green), 0.5 (blue), and 1 (red) ratios of the IL-8 monomer to ND1–38.
  5. 5. 198 Interactions of Interleukin-8 with CXCR1 and 15N chemical shifts. With increasing concentra- tions 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 3–5 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 Fig. 3. Interaction of IL-8 with full-length CXCR1. 15N- of CXCR1 have been described previously. 13,18,21edited 1H solution NMR spectra of uniformly 15N-labeled IL-8 in the presence of unlabeled full-length CXCR1 in q=0.1 However, information about the interaction of IL-isotropic bicelles. The molar ratios of CXCR1 to IL-8 monomer 8 with full-length CXCR1 is scarce largely becauseare listed on the right side of their respective spectra. 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 variousand analysis of intermolecular nuclear Overhauser CXCR1 constructs in phospholipid bilayers includ-enhancements observed between IL-8 and the 17- ing the full-length protein. 7,8 This enables us toresidue peptide derived from CXCR1 described study the interactions of IL-8 with full-length andabove. 13 Here, we take advantage of having truncated constructs of CXCR1 in membraneprepared an isotopically labeled polypeptide by environments.bacterial expression corresponding to the N-terminal Figure 3 shows the effects of adding increasing thedomain of CXCR1 to map the binding site using amounts of CXCR1 in bilayers to a q = 0.1 isotropicheteronuclear solution NMR experiments. The bicelle solution containing uniformly 15N-labeledchanges in the spectrum of ND1–38 resulting from IL-8. In the absence of the receptor-containingthe addition of unlabeled IL-8 have the characteris- bilayers, the 15N-edited 1H solution NMR spectrumtics of “fast exchange” on the timescales of the 1H of the amide region has narrow and well-dispersed Fig. 4. Interaction of IL-8 with three constructs of CXCR1 in phospholipid bilayers. 15N chemical shift OS solid-stateNMR spectra of uniformly 15N-labeled IL-8 bound to the constructs of CXCR1 in q = 3.2 aligned bicelles: (a) full-lengthCXCR1; (b) the first transmembrane helix domain of CXCR1 (1TM1–72); (c) N-terminal truncated CXCR1 (NT39–350). Themolar ratio of IL-8 to CXCR1 in each sample was 1:1.
  6. 6. Interactions of Interleukin-8 with CXCR1 199resonances, typical of a small globular protein in 8, 21 and their N-terminal domains have highaqueous solution. As the addition of the receptor sequence homology (Fig. S2). Tryptophan residuesapproaches a 1:1 molar ratio of CXCR1:IL-8, nearly are commonly found near the membrane surface,all signals from labeled IL-8 broaden systematically since the polar amide group and hydrophobic ringand disappear into the baseline, with the exception structure of this amino acid facilitate its localizationof a few signals that have been assigned to residues at the polar/apolar interface. 25 Significantly, signalsnear the N- and C-termini. The result was more from both the backbone and the side chain of thedramatic in lipid bilayers, because with CXCR1 in tryptophan residue in ND1–38 are broadened beyondproteoliposomes at a 1:1 molar ratio with IL-8, all of detection in the presence of lipid bilayers (Fig. 1c),the IL-8 signals disappear as a result of their immo- suggesting that the tryptophan residue may serve asbilization upon binding to the CXCR1-containing an anchor on the membrane surface. The tryptophanbilayers. Refolded CXCR1 prepared by our methods residues located in the N-terminal domain of rabbithas been shown to bind IL-8 with an affinity (Kd of CXCR1, one of which is located in the same position1–5 nM) and to couple to G-protein (EC50 ∼ 1 nM), 7,8 as a tryptophan in the human CXCR1 sequence,which are similar to the values previously reported have been shown to be involved directly inin the literature. 3 membrane interactions. 24 The chemical shift perturbation plot for labeled IL-Critical role of the N-terminal domain of CXCR1 8 in Fig. 2d obtained by the addition of unlabeledfor IL-8 binding ND1–38 shows substantial changes in three regions of the primary sequence. The residues that contrib- Comparisons of 15N chemical shift OS solid-state ute to the binding cleft identified in the three-NMR spectra of uniformly 15N-labeled IL-8 bound dimensional structure of IL-8 were the ones mostto unlabeled full-length CXCR1 and constructs strongly affected by the interaction with ND1–38. Theconsisting of the first transmembrane helix domain central region of the ND1–38 primary sequence(1TM1–72) and the N-terminal truncated (NT39–350) (residues 18–27) was most strongly affected byreceptors in lipid bilayers are shown in Fig. 4. These binding to IL-8. This suggests that ND1–38 mayresults demonstrate that the N-terminal domain of adopt an extended conformation when complexedCXCR1 is mainly responsible for the binding of IL-8. to IL-8. Although the proline residues of ND1–38The OS solid-state NMR signals of IL-8 were intense were not monitored in our experiments, alanine-and well resolved when IL-8 was added to full- scanning studies have shown that the two prolines,length and 1TM1–72 receptors aligned in lipid 21 and 29, as well as Tyr27 contribute to thebilayers, demonstrating that their interaction is interactions with IL-8, suggesting that the hydro-strong enough to immobilize and align IL-8 along phobic characteristics of these residues play roles inwith the receptor at a unique orientation in the binding to the N-terminal domain of CXCR1. 26magnetically aligned bilayers (Fig. 4a and b). As a Many studies of chemokines and their interactionscontrol, no IL-8 signals could be observed in OS with receptors have concluded that one or more ofsolid-state NMR experiments in a sample containing the extracellular loops of the receptors are involved.labeled IL-8 and an unlabeled NT39–350 (Fig. 4c). In particular, alanine-scanning experiments haveSince binding to the receptor is necessary to shown that the third and fourth extracellular loopsimmobilize and align the IL-8, this suggests that of CXCR1 are involved in the binding to IL-8. 19 Anthe binding site is predominantly located in the overall broadening of solution NMR signals of IL-N-terminal region of the receptor. 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 lessDiscussion affected than those of IL-8 in the presence of the full- length receptor (Fig. 3). Two possible reasons for this Comparisons between the solution NMR and difference are that the binding of IL-8 to 1TM1–72 issolid-state NMR spectra of ND1–38 alone and not as tight as for the full-length receptor or that thebound to IL-8 provide information about the binding is as tight as full-length receptor, but theinfluence of the lipid bilayer on interactions of the smaller size of the IL-8 and 1TM1–72 complexN-terminal domain of CXCR1 and IL-8. The N- (∼ 18 kDa) reorients faster than IL-8 and the full-terminal region of CXCR1 determines the specificity length complex (∼ 52 kDa) in isotropic q = 0.1and affinity for IL-8. 22,23 Recently, a 34-residue bicelles. In the case of the N-terminal truncatedpeptide with a sequence corresponding to the N- receptor, the molecular mass of NT39–350 is reducedterminal residues of rabbit CXCR1 was shown to by only 10% compared to the full-length receptor;interact with the membrane surface by monitoring thus, the reduction in rotational correlation time isfluorescence of two tryptophan residues of the unlikely to be sufficient to account for the spectralpeptide. 24 Both human and rabbit CXCR1 receptors changes. It may be that the changes are a manifes-have similar affinity and specificity for human IL- tation of weak interactions of IL-8 to extracellular
  7. 7. 200 Interactions of Interleukin-8 with CXCR1loop regions of the receptor without the contribu- peripheral membrane protein, interacts transientlytions from the missing residues in the N-terminal with the membrane surface and adopts a rela-domain of the receptor. tively well-defined yet still flexible structure that The role of dimerization of IL-8 in binding CXCR1 may contribute to receptor selectivity. Our NMRis not fully understood, but recent studies have data on the N-terminal domain of CXCR1 in theshown that the IL-8 monomer binds to the N- absence and presence of phospholipid bicellesterminal domain of CXCR1 with higher affinity than clearly demonstrate the significant effects of thethe IL-8 dimer. 27,28 We used only the monomeric membrane environment on the structure andform of CXCR1, and in all of our experiments, the dynamics of this domain (Fig. 1). In particular,spectral changes stopped when an approximately the Trp10 side chain is likely to be embedded inequimolar concentration of CXCR1 monomer to the the bilayer.IL-8 monomer was achieved. These results suggest In the second step, after binding to IL-8, the N-that one molecule of CXCR1 binds to one molecule terminal domain dissociates from the membraneof the IL-8 monomer. Since IL-8 exists as a stable surface. Upon interaction with IL-8, the solutionhomodimer in an aqueous solution, it is possible NMR signals of the N-terminal domain that werethat the chemical shift perturbation of IL-8 upon completely broadened out due to the membranebinding to CXCR1 constructs results not only from interaction (step 1) reappeared as a result ofthe direct interaction between them but also from dissociation of the domain from the membranethe dimer-to-monomer transition of IL-8. (Fig. 1d). The complementary OS solid-state NMR It is essential to obtain atomic-resolution structural spectrum of the domain in complex did not yielddetails about how IL-8 interacts with its high-affinity any signals, which also demonstrates that themembrane-embedded receptors in order to under- complex is no longer immobilized by interactionsstand the first step of the complex signaling cascade. with the membrane (Fig. 1b).In the meantime, we interpret the NMR results In the third step, the complex of IL-8 and the N-discussed above in terms of a multistep series of terminal domain rearranges to engage a secondinteractions between IL-8 and CXCR1 with signifi- binding site on the receptor, most likely involvingcant contributions from the phospholipid bilayers one or more extracellular loops (Fig. 2b and e). This(Fig. 5). Thus, we propose that the ternary complex of step might be the trigger for the conformationalIL-8/CXCR1/bilayer is an essential species. changes in the receptor needed to activate secondary In the first step, the N-terminal domain of signaling cascades. This does not exclude theCXCR1, which has many characteristics of a possibility that IL-8 interacts simultaneously with Fig. 5. Model of IL-8 interacting with CXCR1 in membranes. Step 1: The N-terminal domain of CXCR1 (green) isflexible yet structured by interacting with the surface of the membrane, contributing to receptor selectivity. The first halfof the domain is mainly involved in membrane interaction, and Trp10 serves as an anchor on the extracellular side of themembrane. Step 2: The strong interaction between the N-terminal domain and IL-8 dissociates the domain from themembrane surface. Step 3: The N-terminal domain in complex with IL-8 is translated to the second binding site of theextracellular loops, potentially creating a conformational change in CXCR1 for subsequent G-protein activation. Amonomer from the IL-8 dimer structure (Protein Data Bank ID 2IL8) is represented. The residues of IL-8 (12, 17, 20, 44, 48,49, 50, 61, and 62) whose chemical shifts were perturbed significantly by interaction with the N-terminal domain ofCXCR1 are shown as red spheres.
  8. 8. Interactions of Interleukin-8 with CXCR1 201the N-terminal domain and extracellular loops of the (NT39–350), the first transmembrane helix domain ofreceptor. CXCR1 (1TM1–72), and the N-terminal extracellular do- A two-site mechanism of chemokine receptor main of CXCR1 (ND1–38) were expressed, purified, andinteraction in which the N-terminal domain and refolded as described previously. 7,8 The amino acid sequences of the CXCR1 constructs are shown in Support-extracellular loop in the receptor are involved in the ing Information. The amino acid sequence of ND1–38ligand interaction has been proposed based on the substitutes Ser for Cys at position 30 to prevent compli-various structure–function studies reviewed by cations due to intermolecular disulfide bond formation.Rajagopalan and Rajarathnam. 29 Although it is not For the solution NMR experiments, the concentration offully understood how the two-site mechanism IL-8 and ND1–38 polypeptides was 0.1 mM, in 20 mMmediates affinity, selectivity, and activation of the Hepes, at pH 5.5, in 400 μl of 90% H2O/10% 2H2O. Thereceptor, the N-terminal residues of the receptor are protein-containing bicelle samples of IL-8 and ND1–38shown to be essential for both binding affinity and were prepared by dissolving the lyophilized polypeptidesreceptor selectivity. 22 The OS solid-state NMR data directly into premixed solutions containing DMPC andpresented here show that the N-terminal domain of DHPC phospholipids. The lipids were obtained from Avanti Polar Lipids†. The isotropic (q = 0.1) and magnet-CXCR1 is mainly responsible for the strong interac- ically alignable (q = 3.2) samples contain 10% DHPC (w/v)tion with IL-8 (Fig. 4). and 10% DMPC (w/v), respectively. The samples of the It has been proposed that the chemokine N-terminal CXCR1 constructs, except for the soluble ND1–38 poly-“ELR” motif interacts with the extracellular loops of peptide, were prepared from proteoliposome pellets [20%the receptor. 30,31 Recently, the highly dynamic (w/v) lipid] in which 1 mg of the polypeptide wasN-terminus including the ELR motif of the chemokine reconstituted into a solution containing 10 mg of DMPC.SDF-1 has been proposed to play a crucial role in the For the titration experiments, a stock solution of theinteraction with its receptor CXCR4. 32 However, we unlabeled proteins under the same buffer conditions wasdo not observe experimental NMR evidence that the added to the uniformly 15N-labeled proteins so that theN-terminal ELR motif of IL-8 interacts with full-length final molar ratios were 0.25, 0.5, and 1.0. For the OS solid-state NMR experiments, 1 mg of theor N-terminal truncated CXCR1. This may be due to unbound form of uniformly 15N-labeled ND1–38 and IL-differences between the two receptors, or it may 8 were dissolved in 200 μl of a q = 3.2 lipid mixturerequire future studies of the structures and mecha- containing 20% DMPC (w/v) and 20 mM Hepes, atnisms of GPCRs to fully sort out. pH 5.5. The complex was formed by adding 0.6 mg of The interactions between ligands and their mem- uniformly 15N-labeled IL-8 to the unlabeled CXCR1brane-embedded receptors, especially GPCRs, are constructs or 1 mg of labeled ND1–38 to the unlabeled IL-the first step in initiating the complex cascades of 8 in a final molar ratio of 1:1. The pH of the IL-8: 1TM1–72protein interactions known to regulate physiological complex was adjusted to 4.7 to increase the sampleprocesses in mammals. Here, we demonstrate that solubility, while the pH of the other samples was 5.5.the interaction between IL-8 and its receptor, CXCR1,must be analyzed in the context of the phospholipid NMR spectroscopybilayer environment. Solid-state NMR spectroscopyis unique in providing atomic-resolution information The solution NMR experiments were performed atabout membrane proteins and their complexes in 40 °C on a Bruker DRX 600-MHz spectrometer equippedphospholipid bilayers under conditions where signal with 5-mm triple-resonance cryoprobe with z-axistransduction occurs. The resulting NMR data enable gradient. Heteronuclear solution NMR experiments wereus to propose a model for the interactions between performed on uniformly 15 N-labeled or uniformly 13IL-8 and CXCR1 that involve the phospholipid C/ 15N-double-labeled samples with a protein concen- tration of 0.1 mM. One-dimensional 15N-edited 1H NMRbilayer, IL-8, the N-terminal domain of CXCR1, spectra resulted from signal averaging of 128 transients.and residues in inter-helical loops near the C-terminus. Two-dimensional 1H/ 15N HSQC spectra were obtainedIn summary, we conclude that the membrane bilayer on uniformly and selectively 15N-labeled samples. Triple-plays a role that is as important as the structural resonance HNCA and HNCOCA experiments werefeatures of the two protein components in the performed on 13C/ 15N-double-labeled IL-8 and ND1–38interactions of IL-8 and CXCR1 in the first step of for resonance assignments. The chemical shift perturba-transducing biological signals. tions by addition of unlabeled samples were calculated using the equation sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðDyΗ Þ2 + ðDyN = 5Þ2Materials and Methods Dy = 2Sample preparation where ΔδH is the change in the backbone amide proton chemical shift and ΔδN is the change in backbone amide nitrogen chemical shift. IL-8 was expressed and purified as describedpreviously. 22 Full-length CXCR1 and three truncatedconstructs including N-terminal truncated CXCR1 † www.avantilipids.com
  9. 9. 202 Interactions of Interleukin-8 with CXCR1 The solid-state 15N NMR spectra were obtained at 40 °C 7. Park, S. H., Prytulla, S., De Angelis, A. A., Brown,on a 700-MHz Bruker Avance spectrometer. The J. M., Kiefer, H. & Opella, S. J. (2006). High-resolutionhomebuilt 1H/ 15N double-resonance probe used in the NMR spectroscopy of a GPCR in aligned bicelles.experiments had a 5-mm-inner-diameter solenoid coil J. Am. Chem. Soc. 128, 7402–7403.tuned to the 15N frequency and an outer MAGC (modified 8. Casagrande, F., Maier, K., Kiefer, H., Opella, S. J. &Alderman–Grant coil) “low E” coil tuned to the 1H Park, S. H. (2011). Expression and purification of G-frequency. 33 The one-dimensional 15N chemical shift protein coupled receptors for NMR structural studies.NMR spectra were obtained by spin-lock cross-polariza- In Production of Membrane Proteins (Robinson, A. S.,tion with a contact time of 1 ms, a recycle delay of 6 s, and ed.), Wiley-vch, Weinheim, Germany.an acquisition time of 10 ms. Transients (4096) were signal 9. Park, S. H., Casagrande, F., Das, B. B., Albrecht, L.,averaged for each spectrum, and an exponential function Chu, M. & Opella, S. J. (2011). Local and globalcorresponding to line broadening of 100 Hz was applied to dynamics of the G protein-coupled receptor CXCR1.each free induction decay prior to Fourier transformation. Biochemistry, 50, 2371–2380.The NMR data were processed using the programs 10. Clore, G. M., Appella, E., Yamada, M., Matsushima,NMRPipe/NMRDraw. 34 The chemical shift frequencies K. & Gronenborn, A. M. (1989). Determination of thewere externally referenced to 15N-labeled solid ammoni- secondary structure of interleukin-8 by nuclearum sulfate, defined as 26.8 ppm, which corresponds to the magnetic resonance spectroscopy. J. Biol. Chem. 264,signal from liquid ammonia at 0 ppm. 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). 1Acknowledgements H NMR solution structure of an active monomeric interleukin-8. Biochemistry, 34, 12983–12990. This research was supported by grants from the 13. Skelton, N. J., Quan, C., Reilly, D. & Lowman, H.National Institutes of Health and utilized the Biotech- (1999). Structure of a CXC chemokine-receptor frag- ment in complex with interleukin-8. Structure, 7,nology Resource Center for NMR Molecular Imaging 157–168.of Proteins at the University of California, San Diego, 14. De Angelis, A. A., Nevzorov, A. A., Park, S. H.,which is supported by grant P41EB002031. F.C. was Howell, S. C., Mrse, A. A. & Opella, S. J. (2004). High-supported by postdoctoral fellowships from the Swiss resolution NMR spectroscopy of membrane proteinsNational Science Foundation (PBBSP3-123151) and in aligned bicelles. J. Am. Chem. Soc. 126, 15340–15341.the Novartis Foundation, formerly the Ciba-Geigy 15. Park, S. H., De Angelis, A. A., Nevzorov, A. A., Wu, C.Jubilee Foundation. H. & Opella, S. J. (2006). Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles. Biophys. J. 91,Supplementary Data 3032–3042. 16. De Angelis, A. A., Howell, S. C., Nevzorov, A. A. & Supplementary data to this article can be found Opella, S. J. (2006). Structure determination of aonline at doi:10.1016/j.jmb.2011.08.025 membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy. J. Am. Chem. Soc. 128, 12256–12267.References 17. Park, S. H., Marassi, F. M., Black, D. & Opella, S. J. (2010). Structure and dynamics of the membrane- 1. Fernandez, E. J. & Lolis, E. (2002). Structure, function, bound form of Pf1 coat protein: implications of and inhibition of chemokines. Annu. Rev. Pharmacol. structural rearrangement for virus assembly. Biophys. Toxicol. 42, 469–499. J. 99, 1465–1474. 2. Visvader, J. E. & Lindeman, G. J. (2008). Cancer stem 18. Clubb, R. T., Omichinski, J. G., Clore, G. M. & cells in solid tumours: accumulating evidence and Gronenborn, A. M. (1994). Mapping the binding unresolved questions. Nat. Rev., Cancer, 8, 755–768. surface of interleukin-8 complexed with an N-termi- 3. Holmes, W. E., Lee, J., Kuang, W. J., Rice, G. C. & nal fragment of the type 1 human interleukin- Wood, W. I. (1991). Structure and functional expres- 8 receptor. FEBS Lett. 338, 93–97. sion of a human interleukin-8 receptor. Science, 253, 19. Leong, S. R., Kabakoff, R. C. & Hebert, C. A. (1994). 1278–1280. Complete mutagenesis of the extracellular domain of 4. Murphy, P. M. & Tiffany, H. L. (1991). Cloning of interleukin-8 (IL-8) type A receptor identifies charged complementary DNA encoding a functional human residues mediating IL-8 binding and signal transduc- interleukin-8 receptor. Science, 253, 1280–1283. tion. J. Biol. Chem. 269, 19343–19348. 5. Ginestier, C., Liu, S., Diebel, M. E., Korkaya, H., Luo, 20. Lian, L. & Roberts, G. C. K. (1993). Effects of chemical M., Brown, M. et al. (2010). CXCR1 blockade selec- exchange on NMR spectra. In NMR of Macromolecules. tively targets human breast cancer stem cells in vitro A Practical Approach (Roberts, G. C. K., ed.), and in xenografts. J. Clin. Invest. 120, 485–497. pp. 153–182, Oxford University Press, Oxford, UK. 6. Wu, D., LaRosa, G. J. & Simon, M. I. (1993). G protein- 21. Gayle, R. B., III, Sleath, P. R., Srinivason, S., Birks, coupled signal transduction pathways for interleukin- C. W., Weerawarna, K. S., Cerretti, D. P. et al. (1993). 8. Science, 261, 101–103. Importance of the amino terminus of the interleukin-
  10. 10. Interactions of Interleukin-8 with CXCR1 203 8 receptor in ligand interactions. J. Biol. Chem. 268, domain: role of coupled interactions and dynamics. 7283–7289. Biochemistry, 48, 8795–8805.22. Rajagopalan, L. & Rajarathnam, K. (2004). Ligand 29. Rajagopalan, L. & Rajarathnam, K. (2006). Structural selectivity and affinity of chemokine receptor CXCR1. basis of chemokine receptor function—a model for Role of N-terminal domain. J. Biol. Chem. 279, binding affinity and ligand selectivity. Biosci. Rep. 26, 30000–30008. 325–339.23. Prado, G. N., Suetomi, K., Shumate, D., Maxwell, C., 30. Clark-Lewis, I., Schumacher, C., Baggiolini, M. & Ravindran, A., Rajarathnam, K. & Navarro, J. (2007). Moser, B. (1991). Structure–activity relationships of Chemokine signaling specificity: essential role for the interleukin-8 determined using chemically synthe- N-terminal domain of chemokine receptors. Biochem- sized analogs. Critical role of NH2-terminal residues istry, 46, 8961–8968. and evidence for uncoupling of neutrophil chemotax-24. Haldar, S., Raghuraman, H., Namani, T., Rajarath- is, exocytosis, and receptor binding activities. J. Biol. nam, K. & Chattopadhyay, A. (2010). Membrane Chem. 266, 23128–23134. interaction of the N-terminal domain of chemokine 31. Moser, B., Dewald, B., Barella, L., Schumacher, C., receptor CXCR1. Biochim. Biophys. Acta, 1798, Baggiolini, M. & Clark-Lewis, I. (1993). Interleukin- 1056–1061. 8 antagonists generated by N-terminal modification.25. Schiffer, M., Chang, C. H. & Stevens, F. J. (1992). The J. Biol. Chem. 268, 7125–7128. functions of tryptophan residues in membrane pro- 32. Kofuku, Y., Yoshiura, C., Ueda, T., Terasawa, H., teins. Protein Eng. 5, 213–214. Hirai, T., Tominaga, S. et al. (2009). Structural basis of26. Attwood, M. R., Borkakoti, N., Bottomley, G. A., the interaction between chemokine stromal cell- Conway, E. A., Cowan, I., Fallowfield, A. G. et al. derived factor-1/CXCL12 and its G-protein-coupled (1996). Identification and characterisation of an receptor CXCR4. J. Biol. Chem. 284, 35240–35250. inhibitor of interleukin-8: a receptor based approach. 33. Grant, C. V., Yang, Y., Glibowicka, M., Wu, C. H., Bioorg. Med. Chem. Lett. 6, 1869–1874. Park, S. H., Deber, C. M. & Opella, S. J. (2009). A27. Fernando, H., Chin, C., Rosgen, J. & Rajarathnam, K. Modified Alderman–Grant Coil makes possible an (2004). Dimer dissociation is essential for interleukin- efficient cross-coil probe for high field solid-state NMR 8 (IL-8) binding to CXCR1 receptor. J. Biol. Chem. 279, of lossy biological samples. J. Magn. Reson. 201, 87–92. 36175–36178. 34. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G.,28. Ravindran, A., Joseph, P. R. & Rajarathnam, K. (2009). Pfeifer, J. & Bax, A. (1995). NMRPipe: a multidimen- Structural basis for differential binding of the inter- sional spectral processing system based on UNIX leukin-8 monomer and dimer to the CXCR1 N- pipes. J. Biomol. NMR, 6, 277–293.