Probing the role of CXC motif in chemokine CXCL8 for high affinity binding and activation of CXCR1 and CXCR2 receptors.
All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K(i) > 1 μm). Further, CC-CXCL8 failed to mobilize Ca(2+) in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca(2+) in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.
Joseph
Prem Raj B
PR
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas 77555, USA.
Sarmiento
Jose M
JM
Mishra
Anurag K
AK
Das
Sandhya T
ST
Garofalo
Roberto P
RP
Navarro
Javier
J
Rajarathnam
Krishna
K
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D018922:HL-60 Cells; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D056004:Molecular Dynamics Simulation; D009504:Neutrophils / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D023062:Receptors, Interleukin-8A / Q000235:genetics / Q000378:metabolism*; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000378:metabolism*
D016209:Interleukin-8; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D002118:Calcium