NMR Analysis of the Structure, Dynamics, and Unique Oligomerization Properties of the Chemokine CCL27

Abstract

Chemokines have two essential interactions in vivo, with G protein-coupled receptors, which activate intracellular signaling pathways, and with glycosaminoglycans (GAGs), which are involved in cell surface localization and transport. Although it has been shown that chemokines bind and activate their respective G protein-coupled receptors as monomers, many chemokines oligomerize upon GAG binding, and the ability to oligomerize and bind GAGs is required for in vivo function. In this study, we investigated the structure, dynamics, and oligomerization behavior of cutaneous T-cell-attracting chemokine (CTACK, also known as CCL27) by NMR.15 N relaxation and translational self-diffusion rates indicate that CCL27 oligomerizes, but in contrast to many other chemokines that form relatively discrete oligomers, CCL27 transitions between monomer, dimer, and tetramer species over a relatively narrow concentration range. A three-dimensional structure determination was pursued under conditions where CCL27 is primarily dimeric, revealing the standard motif for a chemokine monomer. Analysis of chemical shift perturbations of 1 H-15 N HSQC spectra, relaxation-dispersion experiments, and filtered nuclear Overhauser effects suggest that CCL27 does not adopt a discrete CXC or CC dimer motif. Instead, CCL27 has uncommon oligomerization behavior, where several equilibria involving relatively low affinity interactions between different interfaces seem to be simultaneously at work. However, interaction with heparin avidly promotes oligomerization under conditions where CCL27 is monomeric by itself. We hypothesize that the plasticity in the oligomerization state may enable CCL27 to adopt different oligomeric structures, depending on the nature of the GAG binding partner, thereby providing a mechanism for increased diversity and specificity in GAG-binding and GAGrelated functions.

Exploring Platelet Chemokine Antimicrobial Activity: Nuclear Magnetic Resonance Backbone Dynamics of NAP-2 and TC-1

“…To investigate further the monomer-dimer properties of NAP-2 and TC-1, NMR diffusion studies were conducted using the internal standard dioxane, as has been done for other chemokines (9,20,51). For these experiments, the concentrations and conditions of the chemokine samples were carefully maintained to be consistent with each other for direct comparison, and a dilute protein concentration of 0.1 mM was used to obtain a more observable difference in the dimerization properties of the chemokines.…”

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

“…Previous studies have shown that the CXCL8 dimer is the high-affinity GAG ligand (26) and that, for various CC chemokines, GAG binding and dimerization/oligomerization are coupled (45,46). Chemokine expression in vivo is complex, and both robust expression of multiple chemokines and selective expression of just one chemokine have been observed, depending upon the biological context.…”

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

“…Two x-ray structures of chemokine-disaccharide are known, and most NMR structural studies also report only disaccharide binding, which provides only limited insights (46,47). However, NMR studies have had better success with longer GAGs, but the extent of the structural insights varies among different chemokines and seems to be critically dependent on experimental conditions (21,31,48). Recent studies by Handel and co-workers (21) on CCL27 and Volkman and co-workers (31) on CXCL12 using different oligosaccharides, protein concentrations, protein:GAG ratios, and variants (monomer and dimer) have shown that chemokine structural plasticity, oligomerization properties, GAG length, and binding-induced oligomerization/aggregation/precipitation are intimately coupled.…”