Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain.
Chemokines elicit their function by binding receptors of the G-protein-coupled receptor class, and the N-terminal domain (N-domain) of the receptor is one of the two critical ligand-binding sites. In this study, the thermodynamic basis for binding of the chemokine interleukin-8 (IL-8) to the N-domain of its receptor CXCR1 was characterized using isothermal titration calorimetry. We have shown previously that only the monomer of IL-8, and not the dimer, functions as a high-affinity ligand, so in this study we used the IL-8(1-66) deletion mutant which exists as a monomer. Calorimetry data indicate that the binding is enthalpically favored and entropically disfavored, and a negative heat capacity change indicates burial of hydrophobic residues in the complex. A characteristic feature of chemokine receptor N-domains is the large number of acidic residues, and experiments using different buffers show no net proton transfer, indicating that the CXCR1 N-domain acidic residues are not protonated in the binding process. CXCR1 N-domain peptide is unstructured in the free form but adopts a more defined structure in the bound form, and so binding is coupled to induction of the structure of the N-domain. Measurements in the presence of the osmolyte, trimethylamine N-oxide, which induces the structure of unfolded proteins, show that formation of the coupled N-domain structure involves only small DeltaH and DeltaS changes. These results together indicate that the binding is driven by packing interactions in the complex that are enthalpically favored, and are consistent with the observation that the N-domain binds in an extended form and interacts with multiple IL-8 N-loop residues over a large surface area.
Fernando
Harshica
H
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Nagle
Gregg T
GT
Rajarathnam
Krishna
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011817:Rabbits; D023062:Receptors, Interleukin-8A / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D016209:Interleukin-8; D008024:Ligands; D023062:Receptors, Interleukin-8A