Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction
Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction
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DOI：
10.1002/pro.2172
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发表时间：
2012-12-01
期刊：
影响因子：
8
通讯作者：
Brooun, Alexei
中科院分区：
文献类型：
--
作者：
Cardoso, Rosa;Love, Robert;Brooun, Alexei
异源二聚体HIF-1a（缺氧诱导因子）/HIF-β（也称为ARNT-芳烃核转运蛋白）是细胞对缺氧反应的关键介质。这些单体单元之间的相互作用可以通过小分子在其C-末端异二聚化（PasB）结构域之间的结合界面中的作用来修饰。利用HIF-1a PasB结构域变构腔中存在的几个半胱氨酸残基，我们应用基于半胱氨酸的反应组学热点识别策略定位HIF-1a PasB结构域与ARNT相互作用的关键区域。使用基于质谱的初步筛选策略鉴定化合物5，并显示其与HIF-1a PasB结构域的Cys 255特异性反应。HIF-1a和ARNT的PasB结构域之间相互作用的生物物理表征揭示化合物5与Cys 255的共价结合使HIF-1a和ARNT PasB结构域之间的结合亲和力降低约10倍。HIF-1a-PasB-化合物5缀合物的详细NMR结构分析显示，如HIF-1a/ARNT PasB异二聚体的晶体结构所揭示的，与参与HIF-1a/ARNT PasB结构域相互作用的关键残基相关的HIF-1a中的显著局部构象变化。我们的筛选策略可以应用于其他目标，以确定口袋周围的反应性半胱氨酸适合开发小分子调节剂的蛋白质功能。
The heterodimer HIF-1a (hypoxia inducible factor)/HIF-beta (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1a PasB domain, we applied a cysteine-based reactomics hotspot identification strategy to locate regions of HIF-1a PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1a PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1a and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1a and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1a-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1a associated with key residues involved in the HIF-1a/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1a/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.