Using Structural and Dynamic NMR Analyses to Dissect the Pathologic Function of a Novel Chemokine

Abstract

Chemokines are small, secreted proteins that bind G protein-coupled receptors (GPCRs) to orchestrate cellular chemotaxis in the immune system and beyond. While the ∼50 members of the chemokine family share a remarkably conserved tertiary structure, they still manage to coordinate diverse, non-redundant physiologic and pathologic functions in the human body. It is therefore assumed that more subtle nuances in the structure and dynamics of each chemokine-receptor pair are the key determinants of their biologic function. Here we present the molecular structure of CCL28, an especially intriguing chemokine recently identified to play a major role in asthma pathogenesis, solved by solution-state nuclear magnetic resonance spectroscopy (NMR). The structure reveals that while CCL28 adopts the conserved chemokine tertiary fold, it has a particularly curious structural feature not seen in other chemokines. An intrinsically disordered C-terminal domain of ∼30 residues extends from the globular core of the chemokine, part of which is tethered by a non-conserved disulfide bond. Dynamic NMR experiments including backbone 1H-15N heteronuclear NOE measurements, heteronuclear single quantum coherence (HSQC) titrations, and pulsed field gradient (PFG) diffusion measurements collectively indicate the disordered, dynamic nature of the C-terminal extension. Combined with mutational and in vitro functional studies, our findings suggest that the unique structural and dynamic features of CCL28 may be key determinants of its pathologic function.