Identification and characterisation of an inhibitor of interleukin-8: a receptor based approach

Attwood, Michael R.; Borkakoti, Neera; Bottomley, Gillian A.; Conway, Elizabeth A.; Cowan, I. C.; Fallowfield, Amanda G.; Handa, B.K.; Jones, Philip S.; Keech, Elizabeth; Kirtland, Stephen J.; Williams, Glyn; Wilson, Francis X.

doi:10.1016/0960-894x(96)00331-9

Cited by 20 publications

(23 citation statements)

References 23 publications

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“…All of these residues except Thr18 were also identified as being important from the NMR structural studies. On the other hand, mutational studies using a CXCR1 N‐domain peptide showed that some of the acidic residues, in addition to the apolar residues, are involved in binding to neutrophil receptors [20]. Although the results from the mutational studies of the receptor N‐domain are inconclusive, they do indicate that the binding involves interactions with multiple IL‐8 N‐loop residues over a large surface area.…”

Section: Resultsmentioning

confidence: 99%

Thermodynamic characterization of interleukin‐8 monomer binding to CXCR1 receptor N‐terminal domain

2006

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Chemokines constitute the largest family of proteins that mediate leukocyte recruitment and trafficking [1,2]. They show a remarkable range of receptor selection and function, with some binding a single receptor, some binding multiple receptors, and some binding one receptor with high affinity and others with low affinity [3][4][5][6][7]. Chemokine receptors belong to the superclass of G-protein-coupled receptors (GPCRs), and structure-function studies show that all chemokines bind their receptors using the same two-site mechanism, which involves interaction between the ligand N-loop and the receptor N-terminal domain (N-domain) residues and between ligand N-terminal and receptor extracellular loop residues [5]. The largest sequence difference among chemokines and their receptors is found in the N-loop and N-domain, respectively, suggesting that these residues encode both the specificity and promiscuity of interactions.Interleukin-8 (IL-8, also known as CXCL8) and related neutrophil-activating chemokines (such as Chemokines elicit their function by binding receptors of the G-protein-coupled receptor class, and the N-terminal domain (N-domain) of the receptor is one of the two critical ligand-binding sites. In this study, the thermodynamic basis for binding of the chemokine interleukin-8 (IL-8) to the N-domain of its receptor CXCR1 was characterized using isothermal titration calorimetry. We have shown previously that only the monomer of IL-8, and not the dimer, functions as a high-affinity ligand, so in this study we used the IL-8(1-66) deletion mutant which exists as a monomer. Calorimetry data indicate that the binding is enthalpically favored and entropically disfavored, and a negative heat capacity change indicates burial of hydrophobic residues in the complex. A characteristic feature of chemokine receptor N-domains is the large number of acidic residues, and experiments using different buffers show no net proton transfer, indicating that the CXCR1 N-domain acidic residues are not protonated in the binding process. CXCR1 N-domain peptide is unstructured in the free form but adopts a more defined structure in the bound form, and so binding is coupled to induction of the structure of the N-domain. Measurements in the presence of the osmolyte, trimethylamine N-oxide, which induces the structure of unfolded proteins, show that formation of the coupled N-domain structure involves only small DH and DS changes. These results together indicate that the binding is driven by packing interactions in the complex that are enthalpically favored, and are consistent with the observation that the N-domain binds in an extended form and interacts with multiple IL-8 N-loop residues over a large surface area.Abbreviations ASA, accessible surface area; CXCR1, CXC chemokine receptor 1; GPCR, G-protein-coupled receptor; IL-8, interleukin-8; ITC, isothermal titration calorimetry; N-domain, N-terminal domain; TMAO, trimethylamine N-oxide.

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Section: Resultsmentioning

confidence: 99%

Thermodynamic characterization of interleukin‐8 monomer binding to CXCR1 receptor N‐terminal domain

2006

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“…Although the proline residues of ND 1-38 were not monitored in our experiments, alanine-scanning studies have shown that the two prolines, 21 and 29, as well as Tyr27 contribute to the interactions with IL-8, suggesting that the hydrophobic characteristics of these residues play roles in binding to the N-terminal domain of CXCR1. 26 …”

Section: Discussionmentioning

confidence: 99%

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

Park

Casagrande

Cho

et al. 2011

Journal of Molecular Biology

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

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“…The entire N-domain was observed to be sparingly soluble in solution, and therefore we used two constructs, comprising 23 and 34 residues of the full-length N-domain. Both constructs lacked 10 amino acids on the N-terminal end, and these have been shown not essential for binding (39). In addition, the 34-mer construct contained 11 membrane proximal residues on the C terminus of the N-domain.…”

Section: Design and Synthesis Of N-domain Constructs-mentioning

confidence: 99%

“…Thus, to understand the individual components of the two-site interaction, as well as to understand the coupling between these sites, we need to measure the individual interactions under conditions similar to those seen in the intact receptor. CXCR1 Ndomain fragments inhibit IL-8 binding to the intact receptor with an inhibition constant (K i ) of ϳ20 M (35,39). Because the N-domain is proximal to the membrane interface, we measured the direct binding of the ligand to the N-domain in micelles that mimic the native membrane environment.…”

Section: Ligand Binding Affinities Of N-domain Peptides In Solution Amentioning

confidence: 99%

Ligand Selectivity and Affinity of Chemokine Receptor CXCR1

Rajagopalan

Rajarathnam

2004

Journal of Biological Chemistry

121

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Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (Jdomain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (ϳ1 M versus ϳ20 M) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (ϳ3 M). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the Ndomain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.

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Identification and characterisation of an inhibitor of interleukin-8: a receptor based approach

Cited by 20 publications

References 23 publications

Thermodynamic characterization of interleukin‐8 monomer binding to CXCR1 receptor N‐terminal domain

Thermodynamic characterization of interleukin‐8 monomer binding to CXCR1 receptor N‐terminal domain

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

Ligand Selectivity and Affinity of Chemokine Receptor CXCR1

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