Skip to main content
Log in

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Alexander SP et al (2015) The Concise Guide to PHARMACOLOGY 2015/16: G protein-coupled receptors. Br J Pharmacol 172:5744–5869

    Article  Google Scholar 

  • Baggiolini M (2015) CXCL8-the first chemokine. Front Immunol 6:258

    Article  Google Scholar 

  • Baldwin ET et al (1991) Crystal structure of interleukin 8: symbiosis of NMR and crystallography. Proc Natl Acad Sci USA 88:502–506

    Article  ADS  Google Scholar 

  • Barter EF, Stone MJ (2012) Synergistic interactions between chemokine receptor elements in recognition of interleukin-8 by soluble receptor mimics. BioChemistry 51:1322–1331

    Article  Google Scholar 

  • Bendall L (2005) Chemokines and their receptors in disease. Histol Histopathol 20:907–926

    Google Scholar 

  • Bizzarri C et al (2006) ELR + CXC chemokines and their receptors (CXC chemokine receptor 1 and CXC chemokine receptor 2) as new therapeutic targets. Pharmacol Ther 112:139–149

    Article  Google Scholar 

  • Burg JS et al (2015) Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor. Science 347:1113–1117

    Article  ADS  Google Scholar 

  • Burrows SD et al (1994) Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations. BioChemistry 33:12741–12745

    Article  Google Scholar 

  • Casagrande F, Maier K, Kiefer H, Opella SJ, Park SH (2011) Expression and purification of G-protein coupled receptors for NMR structural studies. In: Production of membrane proteins. Wiley, Weinheim

    Google Scholar 

  • Chaudhuri A et al (2013) Organization and dynamics of the N-terminal domain of chemokine receptor CXCR1 in reverse micelles: effect of graded hydration. J Phys Chem B 117:1225–1233

    Article  Google Scholar 

  • Clark-Lewis I, Schumacher C, Baggiolini M, Moser B (1991) Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J Biol Chem 266:23128–23134

    Google Scholar 

  • Clore GM, Appella E, Yamada M, Matsushima K, Gronenborn AM (1990) Three-dimensional structure of interleukin 8 in solution. BioChemistry 29:1689–1696

    Article  Google Scholar 

  • Clubb RT, Omichinski JG, Clore GM, Gronenborn AM (1994) Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor. FEBS Lett 338:93–97

    Article  Google Scholar 

  • Eigenbrot C, Lowman HB, Chee L, Artis DR (1997) Structural change and receptor binding in a chemokine mutant with a rearranged disulfide: X-ray structure of e38C/C50A IL-8 at 2 Å resolution. Proteins 27:556-566

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Fernando H, Nagle GT, Rajarathnam K (2007) Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain. FEBS J 274:241–251

    Article  Google Scholar 

  • Gayle RB et al (1993) Importance of the amino terminus of the interleukin-8 receptor in ligand interactions. J Biol Chem 268:7283–7289

    Google Scholar 

  • Gerber N, Lowman H, Artis DR, Eigenbrot C (2000) Receptor-binding conformation of the “ELR” motif of IL-8: X-ray structure of the L5C/H33C variant at 2.35 A resolution. Proteins 38:361–367

    Article  Google Scholar 

  • Grasberger BL, Gronenborn AM, Clore MG (1993) Analysis of the backbone dynamics of interleukin-8 by 15N relaxation measurements. J Mol Biol 230:364–372

    Article  Google Scholar 

  • Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925

    Article  Google Scholar 

  • Haldar S, Raghuraman H, Namani T, Rajarathnam K, Chattopadhyay A (2010) Membrane interaction of the N-terminal domain of chemokine receptor CXCR1. Biochim Biophys Acta 1798:1056–1061

    Article  Google Scholar 

  • Hammond ME et al (1996) Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket. J Biol Chem 271:8228–8235

    Article  Google Scholar 

  • Hebert CA, Vitangcol RV, Baker JB (1991) Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem 266:18989–18994

    Google Scholar 

  • Hébert CA et al (1993) Partial functional mapping of the human interleukin-8 type A receptor. Identification of a major ligand binding domain. J Biol Chem 268:18549–18553

    Google Scholar 

  • Helmer D et al (2015) Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex. Rsc Adv 5:25657–25668

    Article  Google Scholar 

  • Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI (1991) Structure and functional expression of a human interleukin-8 receptor. Science 253:1278–1280

    Article  ADS  Google Scholar 

  • Jiang S-J et al (2015) Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1. Scientific Rep 5:18638

    Article  ADS  Google Scholar 

  • Joseph PR, Rajarathnam K (2015) Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain. Protein Sci 24:81–92

    Article  Google Scholar 

  • Joseph PR et al (2010) Probing the role of CXC motif in chemokine CXCL8 for high affinity binding and activation of CXCR1 and CXCR2 receptors. J Biol Chem 285:29262–29269

    Article  Google Scholar 

  • Joseph PR et al (2013) Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins. Biophys J 105:1491–1501

    Article  Google Scholar 

  • Joseph PR, Mosier PD, Desai UR, Rajarathnam K (2015) Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions. Biochem J 472:121–133

    Article  Google Scholar 

  • Kendrick AA et al (2014) The dynamics of interleukin-8 and its interaction with human CXC receptor I peptide. Protein Sci 23:464–480

    Article  Google Scholar 

  • Krieger E et al (2004) A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: support from isothermal fluorescence titrations. Proteins 54:768–775

    Article  Google Scholar 

  • Kufareva I, Gustavsson M, Zheng Y, Stephens BS, Handel TM (2017) What do structures tell us about chemokine receptor function and antagonism? Annu Rev Biophys 46:175–198

    Article  Google Scholar 

  • LaRosa GJ et al (1992) Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity. J Biol Chem 267:25402–25406

  • Leong SR, Kabakoff RC, Hébert CA (1994) Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction. J Biol Chem 269:19343–19348

    Google Scholar 

  • Liou J-W et al. In silico analysis reveals sequential interactions and protein conformational changes during the binding of chemokine CXCL-8 to its receptor CXCR1. PLoS ONE 9(2014): e94178

    Article  ADS  Google Scholar 

  • Lowman HB et al (1997) Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation. Protein Science 6:598–608

    Article  Google Scholar 

  • Mesleh MF, Opella SJ (2003) Dipolar Waves as NMR maps of helices in proteins. J Magn Reson 163:288–299

    Article  ADS  Google Scholar 

  • Möbius K et al (2013) Investigation of lysine side chain interactions of interleukin-8 with heparin and other glycosaminoglycans studied by a methylation-NMR approach. Glycobiology 23:1260–1269

    Article  Google Scholar 

  • Monteclaro FS, Charo IF (1997) The amino-terminal domain of CCR2 is both necessary and sufficient for high affinity binding of monocyte chemoattractant protein 1—Receptor activation by a pseudo-tethered ligand. J Biol Chem 272:23186–23190

    Article  Google Scholar 

  • Murphy PM, Tiffany HL (1991) Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253:1280–1283

    Article  ADS  Google Scholar 

  • Murphy PM et al (2000) International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52:145–176

    Google Scholar 

  • Nasser MW et al (2009) Differential activation and regulation of CXCR1 and CXCR2 by CXCL8 monomer and dimer. J Immunol 183:3425–3432

    Article  Google Scholar 

  • Oswald C et al (2016) Intracellular allosteric antagonism of the CCR9 receptor. Nature 540:462–465

    Article  ADS  Google Scholar 

  • Park SH, Casagrande F, Cho L, Albrecht L, Opella SJ (2011a) Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy. J Mol Biol 414:194–203

    Article  Google Scholar 

  • Park SH et al (2011b) Local and global dynamics of the G protein-coupled receptor CXCR1. BioChemistry 50:2371–2380

    Article  Google Scholar 

  • Park SH et al (2012) Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491:779–783

    Article  ADS  Google Scholar 

  • Park SH et al. (2014) Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA). J Biomol NMR 61(3–4):185–196

    Google Scholar 

  • Park SH, Berkamp S, Radoicic J, De Angelis AA, Opella SJ (2017) Interaction of monomeric interleukin-8 with CXCR1 mapped by proton-detected fast MAS solid-state NMR and intermolecular paramagnetic relaxation enhancement. (Submitted)

  • Prado GN et al (2007) Chemokine signaling specificity: essential role for the N-terminal domain of chemokine receptors. BioChemistry 46:89618968

    Google Scholar 

  • Qin L et al (2015) Structural biology. Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science 347:1117–1122

    Article  ADS  Google Scholar 

  • Rajagopalan L, Rajarathnam K (2004) Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain. J Biol Chem 279:30000–30008

    Article  Google Scholar 

  • Rajarathnam K, Clark-Lewis I, Sykes BD (1995) 1H NMR solution structure of an active monomeric interleukin-8. BioChemistry 34:12983–12990

    Article  Google Scholar 

  • Rajarathnam K, Kay CM, Clark-Lewis I, Sykes BD (1997) Characterization of quaternary structure of interleukin-8 and functional implications. Methods Enzymol 287:89–105

    Article  Google Scholar 

  • Rajarathnam K, Prado GN, Fernando H, Clark-Lewis I, Navarro J (2006) Probing receptor binding activity of interleukin-8 dimer using a disulfide trap. BioChemistry 45:7882–7888

    Article  Google Scholar 

  • Ravindran A, Joseph PR, Rajarathnam K (2009) Structural basis for differential binding of the interleukin-8 monomer and dimer to the CXCR1 N-domain: role of coupled interactions and dynamics. BioChemistry 48:8795–8805

    Article  Google Scholar 

  • Ritchie TK et al (2009) Chapter 11—Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231

    Article  Google Scholar 

  • Rosenkilde MM, Schwartz TW (2004) The chemokine system—a major regulator of angiogenesis in health and disease. APMIS 112:481–495

    Article  Google Scholar 

  • Schwieters CD, Kuszewski JJ, Clore MG (2006) Using Xplor‚ÄìNIH for NMR molecular structure determination. Prog Nucl Magn Reson Spectrosc 48:47–62

    Article  Google Scholar 

  • Skelton NJ, Quan C, Reilly D, Lowman H (1999) Structure of a CXC chemokine-receptor fragment in complex with interleukin-8. Structure 7:157–168

    Article  Google Scholar 

  • Suetomi K, Rojo D, Navarro J (2002) Identification of a signal transduction switch in the chemokine receptor CXCR1. J Biol Chem 277:31563–31566

    Article  Google Scholar 

  • Tan Q et al (2013) Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341:1387–1390

    Article  ADS  Google Scholar 

  • Tian Y, Schwieters CD, Opella SJ, Marassi FM (2015) A practical implicit membrane potential for NMR structure calculations of membrane proteins. Biophys J 109:574–585

    Article  Google Scholar 

  • Williams G et al (1996) Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding. J Biol Chem 271:9579–9586

    Article  Google Scholar 

  • Wu L et al (1996) Discrete steps in binding and signaling of interleukin-8 with its receptor. J Biol Chem 271:31202–31209

    Article  Google Scholar 

  • Wu B et al (2010) Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330:1066–1071

    Article  ADS  Google Scholar 

  • Zheng Y et al (2016) Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540:458–461

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We thank Mitchell Zhao for his participation in the sample preparation and Dr. Andrey Bobkov for assistance with the ITC measurements. This research was supported by Grants P41EB002031, RO1GM066978, R35GM122501, and R35GM118186 from the National Institutes of Health and utilized the Biomedical Technology Resource Center for NMR Molecular Imaging of Proteins at the University of California, San Diego.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stanley J. Opella.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10858_2017_128_MOESM1_ESM.tif

Supplementary Figure 1—1H-15N HSQC spectrum of monomeric IL-8(1-66) after 1 hour incubation in D2O. The five most intense resonances are marked with their residue numbers (TIF 4563 KB)

10858_2017_128_MOESM2_ESM.tif

Supplementary Figure 2—Isothermal Titration Calorimetry experimental results. (A) ITC titration of monomeric IL-8 (1-66) and ND-CXCR1(1-38). N=0.869, KD = 3.5 M, ΔH = −3.2 kcal/mol and ΔS=14.1 cal/mol/deg (B) ITC titration of monomeric IL-8 (1-66) and 1TM-CXCR1 (1-72) reconstituted in MSP1D1ΔH5 nanodiscs N= 0.357, KD = 12.5 M, ΔH=−3.1 kcal/mol and ΔS=11.7 cal/mol/deg Top panel shows experimental data. Bottom panel shows integrated data fitted to extracted values of KD, ΔH and ΔS, after subtraction of titrations of IL-8 in buffer and empty nanodiscs, respectively (TIF 6516 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Berkamp, S., Park, S.H., De Angelis, A.A. et al. Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy. J Biomol NMR 69, 111–121 (2017). https://doi.org/10.1007/s10858-017-0128-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-017-0128-3

Keywords

Navigation