Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and-4 Using Phage Display
Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and-4 Using Phage Display
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DOI：
10.1074/jbc.m114.599233
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发表时间：
2014-11-14
影响因子：
4.8
通讯作者：
Proudfoot, Amanda E. I.
中科院分区：
文献类型：
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作者：
Bonvin, Pauline;Dunn, Steven M.;Proudfoot, Amanda E. I.
背景：趋化因子结合蛋白Evasin-1和-4的选择性不同。结果：通过噬菌体展示，我们确定了Evasin-4的n端区域是与CC趋化因子相互作用的关键。结论：Evasin-1和-4利用不同的结构域结合靶标。意义：噬菌体展示可以快速了解它们的不同选择性，有助于合理设计抑制蛋白。为了阐明由血鼻蜱产生的CC趋化因子结合蛋白Evasin-1和Evasin-4的配体结合表面，我们试图通过噬菌体展示表达Evasin突变体来确定影响其不同趋化因子选择性的关键决定因素。我们首先基于Evasin-1CCL3复合物结构和Evasin-4与CCL3结合的计算机模型设计了丙氨酸突变体。将突变体展示在M13噬菌体颗粒上，ELISA检测其与趋化因子的结合情况。然后将选定的变体作为纯化蛋白生产，并通过表面等离子体共振分析和趋化性抑制来表征。通过确认ph -14和Trp-89对Evasin-1抑制特性的重要性，验证了该方法，并鉴定了第三个关键残基Asn-88。两个氨基酸，Glu-16和tyr19，被鉴定为结合和抑制Evasin-4的关键残基。在平行方法中，我们鉴定了一个克隆（Y28Q/N60D），其与CCL3、CCL5和CCL8的结合明显减少。因此，Evasin-1和-4似乎使用不同的药物载体结合CC趋化因子，主要结合发生在Evasin-1的C端，而不是通过Evasin-4的n端区域。然而，这两种蛋白似乎都靶向趋化因子N端，可能是因为这些结构域是受体信号传导的关键。结果还表明，噬菌体展示可能为小抑制结合蛋白的药效团的快速研究提供了一种有用的方法。
Background: The selectivity profiles of the closely related chemokine-binding proteins Evasin-1 and -4 differ.Results: Using phage display, we identified the N-terminal region of Evasin-4 as key for the interaction with CC chemokines.Conclusion: Evasin-1 and -4 use different domains for target binding. Significance: Phage display allowed rapid insight into their different selectivities, which could aid rational design of inhibitory proteins.To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.