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

Abstract

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.

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

“…Site I comprises the initial interaction between the CXCL8 N-loop residues and the CXCR1 receptor N-terminal residues, while site II involves the CXCL8 N-terminal (ELR motif) and the receptor distant extracellular region. [5][6][7]10,[14][15][16] However, a recent biophysical study performed in lipid bilayers using human full-length CXCR1 and CXCL8 did not fully support such findings, suggesting that the binding process between CXCL8 and human CXCR1 appears to be mainly associated with the N-terminal region of the receptor. 17 Furthermore, using biophysical studies, Ravindran et al 7 proposed that upon receptor engagement, wild-type CXCL8 dissociates to form a CXCL8 monomer/receptor complex.…”

“…Several different mutations or truncations that disrupt the structural order in these regions can lead to formation of dimerization-incapable CXCL8 mutants, which include deletion of Cterminal residues involved in a-helix formation and mutation of the residues within one of the bstrands. [5][6][7][8] Although several studies have characterized the importance of dimerization on the activity of CXCL8 in vitro and in vivo, the molecular and functional consequences of such dimerization are not known in full detail. The dissociation constant (K d ) for CXCL8 dimerization has been reported to be 10 to 20 lM.…”

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…Residues in the N-terminal domain (binding site-I) and in extracellular loops (binding site-II) of CXCR1 interact with IL-8 (13,14,16,(30)(31)(32)(33)35,66,68,69). Specific residues in the N-terminal domain of CXCR1 contribute to both the selectivity and the affinity of the receptor for IL-8.…”

“…The monomer binds to the N-terminal domain of CXCR1 with higher affinity than the dimer (13)(14)(15), and is believed to be the biologically active form of the protein. Modification, mutation, or truncation of residues at the dimer interface stabilize the monomeric form of IL-8 (12,16). The structure of monomeric IL-8 (1-66), obtained by truncation of the last six C-terminal residues, using solution NMR is shown in Fig.…”

“…1 B. Although none of the structures represent complexes of a receptor with one of its native chemokine agonists, the structures of receptors bound to viral chemokines provide information that is complementary to the many mutagenesis and binding studies of their interactions. There have been many solution NMR and other biophysical studies of IL-8 interacting with polypeptides whose sequences correspond to N-terminal residues of CXCR1 (13)(14)(15)(16)(30)(31)(32)(33). The current model for chemokine-receptor interactions involves two binding sites on the receptor.…”