Backbone dynamics of the human CC chemokine eotaxin: Fast motions, slow motions, and implications for receptor binding

Eotaxin is a member of the chemokine family of about 40 proteins that induc e cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is high ly expressed by eosinophils, and it is considered important in the patholog y of chronic respiratory disorders such as asthma. The high resolution stru cture of eotaxin is known. The 74 amino acid protein has two disulfide brid ges and shows a typical chemokine fold comprised of a core of three antipar allel beta-strands and an overlying alpha-helix. In this paper, we report t he backbone dynamics of eotaxin determined through N-15-T-1, T-2, and {H-1} -N-15 nuclear Overhauser effect heteronuclear multidimensional NMR experime nts. This is the first extensive study of the dynamics of a chemokine deriv ed from 600, 500, and 300 MHz NMR field strengths. From the T-1, T-2, and N OE relaxation data, parameters that describe the internal motions of eotaxi n were derived using the Lipari-Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure element s. However, surrounding the core, the regions known to be functionally impo rtant in chemokines show a range of motions on varying timescales. These in clude extensive subnanosecond to picosecond motions in the N-terminus, C-te rminus, and the N-loop succeeding the disulfides. Analysis of rotational di ffusion anisotropy of eotaxin and chemical exchange terms at multiple field s also allowed the confident identification of slow conformational exchange through the "30s" loop, disulfides, and adjacent residues. In addition, we show that these motions may be attenuated in the dimeric form of a synthet ic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.