Abstract：
Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M-1) while EF1 shows a weaker binding (Ka = 105 M-1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox\'s domain-domain interaction for electron transfer.
摘要：
过氧化氢,由双重氧化酶（Duox）产生，是甲状腺激素合成所必需的.Duox激活涉及Ca2+结合其EF-手结构域（EFD），其中包含两个EF手(EF)。在这项研究中,我们使用光谱法表征了截断的EFD，量热法,电泳迁移率,和凝胶过滤，获得其Ca2+结合的热力学和动力学，以及评估相关的构象变化。我们的结果表明，其第二EF手（EF2）表现出强的放热Ca2结合（Ka=107M-1），而EF1表现出弱的结合（Ka=105M-1），导致其带负电荷的残留物被埋葬。与EFD结合的Ca2导致稳定的结构，其熔融温度从67°C移至99°C，并诱导从二聚体到单体形式的结构转变。EF2似乎在其apo形式的二聚体形成中发挥作用，而与Ca2结合的EF1的疏水暴露对于完整形式的二聚体形成至关重要。结果与从Cryo-EM获得的结构一致，这表明在Ca2+结合时具有疏水斑块的EFD的稳定结构对于其Duox的结构域-结构域相互作用对于电子转移至关重要。
