Ca2+ binding shifts dimeric dual oxidase's truncated EF-hand domain to monomer

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 (K_a = 10⁷ M⁻¹) while EF1 shows a weaker binding (K_a = 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.