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首页> 外文期刊>Journal of Molecular Biology >Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy
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Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy

机译：核磁共振波谱法研究磷脂双层中白细胞介素8与人趋化因子受体CXCR1的相互作用

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摘要

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 15N-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.
机译：CXCR1是趋化因子白介素8（IL-8）的受体，它是免疫和炎性反应的介体。 CXCR1在策略上位于细胞膜中，以高亲和力与IL-8结合，随后将信号跨膜双层转导至G蛋白激活的第二信使系统。在这里，我们描述了脂质环境中IL-8与人类CXCR1之间相互作用的NMR研究。通过在细菌中表达，随后纯化和重折叠，将CXCR1的功能性全长和截短构建体以及全长IL-8进行15N标记。通过IL-8和CXCR1上分配的共振的特定化学位移扰动来鉴定负责IL-8和CXCR1 N末端结构域之间相互作用的残基。当以1：1摩尔比添加脂质双层中的CXCR1时，q = 0.1各向同性双细胞中IL-8的溶液NMR信号完全消失，表明与含受体双层的结合固定了IL-8（在〜10 5 Hz上）时间尺度），并扩大了信号范围，使其无法检测。来自IL-8的相同溶液NMR信号受脂质双层中N端截短的CXCR1添加的影响较小，表明CXCR1的N端结构域主要负责与IL-8的结合。相互作用足够紧密以将IL-8与受体固定在磷脂双层中，并且相互作用足够特异性以在取向的样品固态NMR光谱中产生对齐良好的样品。在各种CXCR1构建体的存在下，IL-8的溶液NMR和固态NMR研究相结合，使我们能够为多步结合过程提供模型。

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