Structural and functional characterization of CC chemokine CCL14.
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.
Blain
Katherine Y
KY
Structural Biology Laboratory, The Salk Institute, La Jolla, California 92037, USA.
Kwiatkowski
Witek
W
Zhao
Qinghai
Q
La Fleur
David
D
Naik
Chethana
C
Chun
Tae-Wook
TW
Tsareva
Tatiana
T
Kanakaraj
Palanisamy
P
Laird
Michael W
MW
Shah
Rutul
R
George
Lisa
L
Sanyal
Indra
I
Moore
Paul A
PA
Demeler
Borries
B
Choe
Senyon
S
D001665:Binding Sites; D020013:Calcium Signaling / Q000187:drug effects*; D019742:Chemokines, CC / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D019713:Receptors, CCR5 / Q000819:agonists* / Q000378:metabolism; D015728:Receptors, HIV / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000187:drug effects / Q000821:virology; D014461:Ultracentrifugation; D053586:Virus Internalization / Q000187:drug effects
C097720:CCL14 protein, human; D019742:Chemokines, CC; D010446:Peptide Fragments; D019713:Receptors, CCR5; D015728:Receptors, HIV; D010450:Endopeptidases