Structures of Human CCL18, CCL3, and CCL4 Reveal Molecular Determinants for Quaternary Structures and Sensitivity to Insulin-Degrading Enzyme

Abstract

CC chemokine ligands (CCL) are 8-14 kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small angle X-ray scattering (SAXS) analyses. Our data shows that CCL18 adopts an α-helical conformation at its N-terminus that weakens its dimerization, explaining CCL18’s preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4, but is replaced by lysine in human CCL18. Our structural analyses suggest that a proline 8 to alanine mutation stabilizes a type I β-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme (IDE) degrades CCL3 and CCL4 but not CCL18. Our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function.

Crystallographic Structure of Truncated CCL21 and the Putative Sulfotyrosine-Binding Site

“…2A), nor was it established between monomers from adjacent asymmetric units in the crystal-packing interface. Additionally, the proline in the N-terminus that has been suggested to promote CC chemokine dimerization is absent in CCL21 (23) . Convincingly, Love et al showed through sedimentation equilibrium centrifugation that full-length CCL21 is monomeric (6) .…”

Structural basis for oligomerization and glycosaminoglycan binding of CCL5 and CCL3

“…The expression of thioreduxin-tagged CCL5, CCL5(4-68), and CCL3 in Escherichia coli, the removal of thioreduxin-tag by enterokinase or tobacco etch virus (TEV) protease, and the purification of these chemokines by Ni-NTA, source-Q, and heparin columns are done similar to methods described previously (14,47). The octasaccharide heparin MLZ-8f was synthesized as described (35).…”

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes

“…An unexplained exception is Aβ 1-40 , the rate of which was increased by approximately twofold by phytic acid. In crystal structures of IDE with bound peptides (28,(35)(36)(37)(38)(39), larger substrates are seen to make contacts at both the active site and the distal site (which mediates activation by peptides). It is possible that activity is not stimulated for large peptides because they themselves activate in a manner similar to polyanions, particularly because their hydrolysis rates are often higher than those for small peptides.…”