Structural and Functional Characterization of CC Chemokine CCL14^,

Abstract

CC chemokine ligand 14, CCL14, is a human CC chemokine that is of recent interest because of its natural ability, upon proteolytic processing of the first eight NH2-terminal residues, to bind to and signal through the human immunodeficiency virus type-1 (HIV-1) co-receptor, CC chemokine receptor 5 (CCR5). We report X-ray crystallographic structures of both full-length CCL14 and signaling-active, truncated CCL14 [9-74] determined at 2.23 and 1.8 A, respectively. Although CCL14 and CCL14 [9-74] differ in their ability to bind CCR5 for biological signaling, we find that the NH2-terminal eight amino acids (residues 1 through 8) are completely disordered in CCL14 and both show the identical mode of the dimeric assembly characteristic of the CC type chemokine structures. However, analytical ultracentrifugation studies reveal that the CCL14 is stable as a dimer at a concentration as low as 100 nM, whereas CCL14 [9-74] is fully monomeric at the same concentration. By the same method, the equilibrium between monomers of CCL14 [9-74] and higher order oligomers is estimated to be of EC1,4 = 4.98 microM for monomer-tetramer conversion. The relative instability of CCL14 [9-74] oligomers as compared to CCL14 is also reflected in the Kd's that are estimated by the surface plasmon resonance method to be approximately 9.84 and 667 nM for CCL14 and CCL14 [9-74], respectively. This approximately 60-fold difference in stability at a physiologically relevant concentration can potentially account for their different signaling ability. Functional data from the activity assays by intracellular calcium flux and inhibition of CCR5-mediated HIV-1 entry show that only CCL14 [9-74] is fully active at these near-physiological concentrations where CCL14 [9-74] is monomeric and CCL14 is dimeric. These results together suggest that the ability of CCL14 [9-74] to monomerize can play a role for cellular activation.

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

“…5, C and D). When the experiment was repeated on a 2.0 mM CCL27 sample, similar results were observed, with additional signals from the first ␤-strand and 3 10 helix that were not present at the higher concentration (supplemental Fig. 4).…”

“…The residues that experienced a change in chemical shift corresponded to several regions of the protein, including Leu-2, Leu-3, Ala-8, Thr-11, and Leu-13 in the N terminus, Ser-19, Asp-20, and Leu-22 in the 3 10 helix, Gln-28 and Leu-31 in the first ␤-strand, Ala-34 to Asp-37 and His-39 to Gln-41 in the 30s loop, Ala-48 to Gln-49 in the 40s loop, Ile-54 in the third ␤-strand, Leu-61, Trp-64, and His-67 to Leu-72 from the C-terminal ␣-helix, and Gly-74, Leu-76, and Lys-78 from the C terminus. The histogram in Fig.…”

“…As expected, CCL2 shows some of the most significant changes in the N terminus, in and around the region forming the small ␤-strand between the monomeric subunits in the dimer structure. The other highly shifted regions include the 3 10 helix, leading into the first ␤-strand, the second ␤-strand leading into the 40s loop and the third ␤-strand, where a disulfide bond connects Cys-52 to Cys-12 in the N terminus. A comparison of the two histograms in Fig.…”

“…Chemokines/receptors have been classified into four subfamilies (CC, CXC, CX3C, and XC) based on the relative positions of conserved cysteine residues in the N termini of the ligands. Structures of many chemokines have been solved and reveal a highly conserved tertiary motif consisting of a disordered N-terminal region followed by a 3 10 helix, three antiparallel ␤-strands, and a C-terminal ␣-helix (10 -13). Despite their similar monomeric structures, multiple dimeric and tetrameric structures have been reported.…”

Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

“…insight.86660DS1) that were not previously identified in proteomics experiments performed with the same platform and for which antibodies to their cellular receptors were available if an ELISA kit was not available. Two lead proteins emerged: (i) CD146, a cell adhesion molecule expressed on ECs particularly during inflammation (18) and on a subset of activated T cells (20, 22, 34), allowing their entry into the intestine through homophilic CD146-CD146 interactions (23, 24); and (ii) CCL14 that binds to the T cell chemokine receptor CCR5 (25, 26), which is known to facilitate T cell infiltration into the intestine (29, 30) and for which trafficking can be blocked by a CCR5 small molecule inhibitor in GI-GVHD patients (32). …”