Solution Structure of Eotaxin, a Chemokine That Selectively Recruits Eosinophils in Allergic Inflammation

Abstract

The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH < 5 and low ionic strength, eotaxin was found to be predominantly a monomer. The three-dimensional structure of the eotaxin monomer solved at pH 5.0 revealed that it has a typical chemokine fold, which includes a 3-stranded ␤-sheet and an overlying ␣-helix. Except for the N-terminal residues (residues 1-8), the core of the protein is well defined. The eotaxin structure is compared with the chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1); eotaxin binds only CC chemokine receptor CCR3, whereas RANTES binds many receptors including CCR3, and MCP-1 binds a distinct receptor, CCR2. The RMSD of the eotaxin ensemble of structures with the RANTES average minimized monomeric subunit is 5.52 ؎ 0.87 Å over all backbone atoms and 1.14 ؎ 0.09 Å over backbone atoms of residues 11-28 and 34 -65. The most important difference between the structures is in the N-terminal residues that are unstructured in eotaxin but structured in RANTES and MCP-1. Several residues in the loop region of RANTES show similar packing in eotaxin (residues 11-17). As the N-terminal and loop regions have been shown to be critical for receptor binding and signaling, this structure will be useful for determining the basis for CCR3 selectivity of the eotaxin.Eotaxin (1, 2) is a member of the chemoattractant cytokine (chemokine) family of proteins that mediates the migration and accumulation of eosinophils at sites of parasitic infection and allergic response (3-5). The chemokines play a pivotal role in the control of leukocyte chemotaxis and represent attractive targets for the blockade of inflammatory disease progression. Eotaxin belongs to the CC chemokine family which is distinguished by the presence of two adjacent cysteines close to the N terminus (6). The CC chemokines activate a wide range of leukocytes including eosinophils, basophils, monocytes, dendritic cells, and T lymphocytes. Chemokine activities are mediated through the binding and activation of seven transmembrane helix G-coupled receptors (6), and eight distinct functional CC chemokine receptors have been identified (6 -9). Their broad leukocyte distribution coupled with a lack of CC chemokine specificity means there is generally considerable redundancy among the CC chemokine family. However, the cloning and characterization of eotaxin and the related protein eotaxin-2 (10) and its receptor CCR3 1 (11) has revealed its unique and most important feature, a high selectivity for eosinophils (1, 2). Eotaxin has been implicated as the predominant chemokine in the recruitment of eosinophils and the eosinophil-mediated tissue damage associated with asthma. Thus eotaxin is a possible target for anti-asthmatic drugs.The three-di...

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

“…Lkn-1 is a truncated form of MIP-1ˇ, with the N-terminal 24 amino acids deleted. Furthermore, when the primary structures of the five chemokines used in this study are compared it is apparent that MIP-1 § , MCP-3, RANTES and Lkn-1 have between seven and ten amino acids N-terminal to the first two cysteine residues (the CC motif), as opposed to the 31 residues of MIP-1ˇ [25]. Interestingly, mutational analyses of MCP-3 and RANTES have shown that the residues N-terminal to the CC motif are critical for the effective induction of THP-1 monocyte cells but not for receptor binding [26].…”

“…This binding step is followed by contact of the N-terminal residues of the chemokine with the receptor. The N-terminal region is considered to mediate activation of the receptor into a conformation that is able to functionally interact with effectors [25]. It is therefore tempting to speculate that the long N-terminal region of MIP-1ˇis unable to activate CCR1 as efficiently as do other agonists, despite being able to bind the receptor with high affinity due to its structural homology to other CC chemokines in the N-loop region.…”

Use of Sulfated Linked Cyclitols as Heparan Sulfate Mimetics to Probe the Heparin/Heparan Sulfate Binding Specificity of Proteins

“…In contrast, none of the sulfated compounds tested, including PI-88, could block eotaxin binding to heparin. Eotaxin also exists as a homodimer (37), so it is likely that the appropriately spaced sulfated motifs were not present in the limited family of sulfated molecules tested. Sulfated cyclitol linker length was also critical for LpL binding inhibition, although in this case a very short spacer was required for optimal inhibitory activity.…”

Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin

“…The receptor binding and activation data are summarized in Table I. DISCUSSION Previous chemokine mutational studies, and their interpretation according to the prevailing two-step model for chemokine receptor activation, have suggested a separation of function between the N-loop and N-terminal regions of chemokines (21,(23)(24)(25). The N-loop is proposed to be important for providing the initial binding energy, whereas the N-terminus is suggested to be required for triggering receptor activation subsequent to binding.…”

“…Extensive mutational studies of several chemokines have led to a proposed "two-step" model for receptor interaction in which the N-loop of the chemokine initially binds to the receptor then the N-terminal region of the chemokine docks with the receptor to induce a conformational change, resulting in receptor activation (21,(23)(24)(25). Recent papers describing the binding of receptor-derived peptides to a groove located between the Nloop and ␤2-␤3 hairpin of their cognate chemokines (interleukin-8, fractalkine, eotaxin, and eotaxin-2) have reinforced the importance of the N-loop region for receptor binding (22, 26 -28).…”