Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy.
CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly (15)N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q=0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the receptor-containing bilayers immobilizes IL-8 (on the ~10(5) Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0307, USA.
Sang Ho
SH
Park
Fabio
F
Casagrande
Leah
L
Cho
Lauren
L
Albrecht
Stanley J
SJ
Opella
D001665:Binding Sites; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008051:Lipid Bilayers; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D023062:Receptors, Interleukin-8A / Q000378:metabolism*
D016209:Interleukin-8; D008051:Lipid Bilayers; D010743:Phospholipids; D023062:Receptors, Interleukin-8A