Structural basis for oligomerization and glycosaminoglycan binding of CCL5 and CCL3

Abstract

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs along with other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer-dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions.signal transduction | CC chemokine | protein oligomerization | glycosaminoglycan | X-ray crystallography T he CC chemokines are a 28-member family of 8-to 14-kDa small-molecular-weight (MW) chemotactic cytokines with crucial roles in inflammation and infection (1, 2). Chemokine oligomerization and their interaction with glycosaminoglycans (GAGs), polysaccharides that are either free or attached to proteoglycans mostly on cell surface or in the extracellular matrix, is a coupled process that play key roles in chemokine functions (3-8). These include, but are not limited to, protection from proteolysis, regulation of chemotactic/haptatactic gradients to guide cell migration, transcytosis of chemokines across cells, and presentation to surface receptors of target cells, particularly under flow conditions. Most CC chemokines readily dimerize by themselves and form higher-MW complexes in the presence of GAGs (4). CC chemokine ligand 3 (CCL3) (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) are CCR5 ligands that are involved in diverse proinflammatory responses and are targeted for therapeutic innovations for human diseases including cancer, cardiovascular diseases, and HIV infection (9-12). Unlike other CC chemokines, these chemokines reversibly self-assemble into high-MW oligomers, up to >600 kDa in size (13,14). The pre...

Two glycosaminoglycan-binding domains of the mouse cytomegalovirus-encoded chemokine MCK-2 are critical for oligomerization of the full-length protein

“…The 30s loop was shown to establish only complementary GAG-interactions in CCL5 (56) and it was proposed to be important for the CX3CL1-GAG interaction; however, this was not demonstrated experimentally (57). On the other hand, the 50s loop has been shown to be critical for the GAG-binding of members of other chemokines subgroups and of CC chemokine oligomers (58,59). Furthermore, we found that the N-terminus of the rm131 chemokine domain plays a more limited role in GAG-binding than it does for other CC chemokines.…”

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…Some chemokines form higher order oligomers (Fig. 1e) (74), many of which utilize both CC and CXC dimerization interfaces (72, 89). …”

CCL3 promotes germinal center B cells sampling by follicular regulatory T cells

“…In that case, to preserve local CCL3 gradient around migrating GC B cell, secreted CCL3 would have to be rapidly internalized and removed by other cells within GCs. Alternatively to the first model, CCL3 chemokine secreted by GC B cells could be transiently immobilized on the surface of selected GC B cells in association with chemokine-binding glycosaminoglycans (Liang, Triandafillou et al 2016) and may serve to stabilize very transient probing interactions of GC B cells with Tfr cells that are beyond the resolution capabilities of intravital 2P microscopy. Both of these models can lead to decreased efficiency in productive sampling of CCL3 KO B cells by Tfr cells.…”