4.7 Article

Interactions of Interleukin-8 with the Human Chemokine Receptor CXCR1 in Phospholipid Bilayers by NMR Spectroscopy

期刊

JOURNAL OF MOLECULAR BIOLOGY
卷 414, 期 2, 页码 194-203

出版社

ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2011.08.025

关键词

membrane protein; solid-state NMR; lipid bilayer; CXCR1; IL-8

资金

  1. National Institutes of Health
  2. Swiss National Science Foundation [PBBSP3-123151]
  3. Novartis Foundation
  4. [P41EB002031]
  5. Swiss National Science Foundation (SNF) [PBBSP3-123151] Funding Source: Swiss National Science Foundation (SNF)

向作者/读者索取更多资源

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 N-15-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 similar to 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. (C) 2011 Elsevier Ltd. All rights reserved.

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