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首页> 外文期刊>Biochemistry >Protein Flexibility of the alpha-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation
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Protein Flexibility of the alpha-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation

机译：蛋白质柔韧性依赖于依赖依赖性氧酶因子抑制HIF-1：对底物结合，催化和调节的影响

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摘要

Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) alpha-ketoglutarate-dependent oxygenase that is a key hypoxia (low p(O2)) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming alpha-ketoglutarate, a protein substrate that is hydroxylated, and O-2, understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting. The primary substrate of FIH is the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor la (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and alpha KG binding, finding that (M+alpha KG) binding significantly stabilized the cupin barrel core of FIH as evidenced by enhanced thermal stability and decreased protein dynamics as assessed by global amide hydrogen/deuterium exchange mass spectrometry and limited proteolysis. Confirming predictions of the consensus mechanism, (M+alpha KG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence. The decreased protein dynamics caused by (M+alpha KG) enforces a sequentially ordered substrate binding sequence in which alpha KG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both alpha KG and the prime substrate. A consequence of the correlation between dynamics and alpha KG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, alpha KG, and inhibitor.
机译：蛋白质动力学对于机械订购的酶至正确的序列与它们的基质结合并进行催化来关键。因子抑制HIF-1（FIH）是依赖于α-酮基的依赖性氧酶，其是人类中的关键缺氧（低P（O2））传感器。由于这些缺氧感测酶遵循多钠化学机制消耗α-酮戊酸，羟基化的蛋白质基质和O-2，了解蛋白质柔性和底物结合的顺序可以有助于开发选择性靶向的策略。 FIH的初级基质是缺氧诱导因子La（HIF）的C-末端转移结构域（CTAD），其在ASN803的侧链上羟化。我们评估了与金属和αkg结合的蛋白质柔性的变化，发现（m +αkg）结合显着稳定了FIH的柴蛋白桶芯，如通过增强的热稳定性和通过全球酰胺氢气/氘交换评估的蛋白质动态减少质谱和有限的蛋白水解。确认共有机制的预测（M +αkg）增加了通过滴定监测内在色氨酸荧光测量的CTAD对CTAD的亲和力。由（m +αkg）引起的蛋白质动态降低强制依次有序的底物结合序列，其中αkg在ctad之前结合，表明选择性抑制可能需要靶向αkg和素底物的结合位点的抑制剂。动力学和αkg结合之间的相关性的结果是，所有相关配体必须包括在基于结合的抑制剂筛选中，如通过M，αkg和抑制剂的测试所示。

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