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

  • MATTHEW P. CRUMP (a1), LEO SPYRACOPOULOS (a2), PIERRE LAVIGNE (a1), KEY-SUN KIM (a3), IAN CLARK-LEWIS (a4) and BRIAN D. SYKES (a1) (a2)...

Abstract

Eotaxin is a member of the chemokine family of about 40 proteins that induce cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is highly expressed by eosinophils, and it is considered important in the pathology of chronic respiratory disorders such as asthma. The high resolution structure of eotaxin is known. The 74 amino acid protein has two disulfide bridges and shows a typical chemokine fold comprised of a core of three antiparallel β-strands and an overlying α-helix. In this paper, we report the backbone dynamics of eotaxin determined through 15N-T1, T2, and {1H}-15N nuclear Overhauser effect heteronuclear multidimensional NMR experiments. This is the first extensive study of the dynamics of a chemokine derived from 600, 500, and 300 MHz NMR field strengths. From the T1, T2, and NOE relaxation data, parameters that describe the internal motions of eotaxin were derived using the Lipari–Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure elements. However, surrounding the core, the regions known to be functionally important in chemokines show a range of motions on varying timescales. These include extensive subnanosecond to picosecond motions in the N-terminus, C-terminus, and the N-loop succeeding the disulfides. Analysis of rotational diffusion anisotropy of eotaxin and chemical exchange terms at multiple fields 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 synthetic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.

Copyright

Corresponding author

Reprint requests to: Brian D. Sykes, The Protein Engineering Network of Centres of Excellence (PENCE) and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada; e-mail: brian.sykes@ualberta.ca.

Keywords

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

  • MATTHEW P. CRUMP (a1), LEO SPYRACOPOULOS (a2), PIERRE LAVIGNE (a1), KEY-SUN KIM (a3), IAN CLARK-LEWIS (a4) and BRIAN D. SYKES (a1) (a2)...

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