Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3.
The CC chemokine eotaxin plays a predominant role in eosinophil trafficking in vivo by specifically activating the chemokine receptor CCR3. We have screened a series of synthetic peptides corresponding to extracellular regions of CCR3 for their ability to bind eotaxin. A peptide corresponding to the N terminus of CCR3 (CCR3-(1-35)) bound to eotaxin with a dissociation constant of 80 +/- 38 micrometer. However, linear or cyclic peptides derived from the first and third extracellular loops of CCR3 did not bind to eotaxin. Linear and cyclic peptides derived from the second extracellular loop precipitated upon addition of eotaxin. (1)H-(15)N correlation NMR spectroscopy indicated that an extended groove in the eotaxin surface, whose edges are defined by the N-loop, 3(10)-helical turn, and beta(2)-beta(3) hairpin, is the most likely binding surface for CCR3-(1-35). NMR assignments for CCR3-(1-35) were obtained using two-dimensional and three-dimensional homonuclear NMR experiments. (15)N-Filtered TOCSY spectra indicated that the central region of CCR3-(1-35), surrounding the DDYY sequence, is involved in the interaction with eotaxin. This was supported by the observation that a truncated N-terminal peptide (CCR3-(8-23)) binds to eotaxin with a dissociation constant of 136 +/- 23 micrometer, only slightly weaker than the full-length N-terminal peptide. Taken together with previous studies, these results suggest that interactions between the N-loop/beta(3) regions of chemokines and the N-terminal regions of their receptors may be a conserved feature of chemokine-receptor complexes across the CC, CXC, and C chemokine subfamilies. However, the low affinity of the interactions observed in these studies suggests the existence of additional binding regions in both the chemokines and the receptors.
Ye
J
J
Department of Chemistry, Indiana University, Bloomington, Indiana 47405-0001, USA.
Kohli
L L
LL
Stone
M J
MJ
D000595:Amino Acid Sequence; D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000737:chemistry*
D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines; D010446:Peptide Fragments; D054397:Receptors, CCR3; D019707:Receptors, Chemokine