New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
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DOI：
10.1371/journal.pone.0031298
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发表时间：
2012-02-20
期刊：
影响因子：
3.7
通讯作者：
Pfeifer, Sven
中科院分区：
文献类型：
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作者：
Hoffmann, Andreas;Kovermann, Michael;Pfeifer, Sven
已经采用了多种方法来产生非抗体支架的结合蛋白。利用人类泛素的β-产生蛋白质泛素的β表，我们获得了与肿瘤坏死因子（TNF）-Alpha的结合蛋白。该验证的药理学靶蛋白的生物活性形式是一种非共价连接的同型均方体。这种结构特征导致观察到有关TNF-Alpha结合分子的结合模式的某种异质性，例如单体/三聚体特异性。我们分析了由核糖体显示选择的基于泛素的TNF-Alpha粘合剂，特别关注其相互作用方式。使用酶联免疫吸附测定法，观察到与纳摩尔亲和力与TNF-α的特异性结合。在等温滴定量热法中，我们获得了有关亲和力的可比结果，并检测到与一个泛素衍生的结合分子结合一个TNF-Alpha trimer的放热反应。使用NMR光谱和其他分析方法可以证实1：3化学计量。详细的结合分析表明，这种相互作用受洗涤剂Tween-20的影响。以前，仅报道了这种现象仅针对另一种类型的替代支架衍生的结合蛋白（设计的端蛋白重复蛋白）未进行进一步研究。正如尺寸排除色谱和NMR光谱法所证明的那样，洗涤剂的存在显着提高了关联率。由于已知TNF-Alpha的特殊结构是由洗涤剂调节的，因此对公认的表位的访问被指示受到靶蛋白内构象转变的限制。我们的结果表明，泛素衍生的结合蛋白靶向TNF-Alpha上的新表位，这与TNF-Alpha中和抗体识别的表位不同。
A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins - designed ankyrin repeat proteins - without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.