Structural and agonist properties of XCL2, the other member of the C-chemokine subfamily

Abstract

Known for its unusual metamorphic native state structure, XCL1 has been the focus of most efforts to elucidate the structural, functional, and physiological properties of chemokines in the C subfamily. By comparison, its closely related paralog XCL2 remains virtually uncharacterized. Based on the importance of the chemokine N-terminus in receptor activation, it was hypothesized that two amino acid differences in XCL2 would alter its agonist activity relative to XCL1 for their shared receptor XCR1. This present study reveals several properties of XCL2 that were unexamined until now. Structurally, XCL1 and XCL2 are very similar, exchanging between the monomeric chemokine fold and an unrelated dimeric state under physiological NaCl and temperature conditions. Ca2+ flux, chemotaxis, and heparin binding assays showed that the monomer form of XCL2 is responsible for G protein-coupled receptor activation while the dimeric form is important for GAG binding. Despite their high structural similarity, XCL2 displays a slightly higher affinity for heparin than XCL1. Because their in vitro functional profiles are virtually identical, distinct physiological roles for XCL1 and XCL2 are probably encoded at the level of expression.

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

“…Data for WT XCL1 and CC3 are shown for comparison and correlate with data from a previous publication. 15 NaCl concentrations at which each XCL1 variant eluted from heparin Sepharose were subtracted from the W55D elution concentration (920 mM) and are reported as Δ[NaCl] H values (Table 1 and Figure 3D). …”

“…4 Protein purification was conducted as previously described 15 with minor modifications. Upon elution from Ni-NTA resin, W55D proteins were refolded by dialysis into warmed (37 °C) refolding buffer consisting of 4 L of 20 mM Tris (pH 8.0), 10 mM cysteine, and 0.5 mM cystine.…”

“…15,17,18 Briefly, ~200 μ g of each purified protein was individually loaded onto 1 mL HiTrap SP-Sepharose HP and heparin HP columns (GE Healthcare). A Shimadzu Prominence HPLC system was used to run the columns with a 0 to 1 M NaCl gradient in 20 mM phosphate (pH 6.0) buffer over 90 min.…”

New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model

“…Chemokines of the C family, XCL1 and XCL2, have recently been shown to exist in a monomer-dimer equilibrium, unusually requiring complete protein unfolding. XCL dimers adopt a novel dimer conformation that also creates a six-stranded β-sheet [133, 134]. CX3CL1, the only member of the CX3C family, dimerizes in a similar manner to that of CC chemokines [135].…”

“…Likewise, GAG binding can favor dimer formation as demonstrated for CCR2-binding chemokines [141], CXCL8 [144, 145], CXCL12 [146–149], XCL1 and XCL2 [133, 134]. Oligomerization has also been shown to increase GAG affinity by creating a more extensive surface for interactions [128].…”