Dual Oxidase (Duox) is a member of the NADPH oxidase (Nox) family responsible for producing H₂O₂ in the thyroid gland and immune cells. It has been shown that Ca²⁺ plays a crucial role in the activation of Duox through its two self-contained putative EF hands (EFs); but its Ca²⁺ binding properties have not yet been reported. Here we prepared a minimal truncated EFD (aa 808-902) and determined its Ca²⁺ binding. Ca²⁺binding induces a conformational change by exposing hydrophobic patches and burying negatively charged residues, as observed in ANS fluorescence and native gel electrophoresis. Using isothermal titration calorimetry, we determined the Ca²⁺binding affinities (Ka) to be on the order of 105 and 107 M-1 for EF1 and EF2, respectively. The strong binding to EF2 is due to the slow dissociation of Ca²⁺ (1.44 s-1) determined by a stopped-flow device. Ca2+ binding also enhanced the protein stability, as determined by differential scanning calorimetry. Interestingly, size exclusion chromatography revealed that the truncated apo-EFD exists as a dimer, and Ca²⁺ binding shifts the oligomer to monomer. It appears that Ca+-unbound EF2 and Ca2+-bound EF1 help stabilize dimer formation. We also expanded our study to encompass the full- length EFD fused with maltose binding protein, and it exhibited a response to Ca²⁺ similar to that of the truncated version. The response included changes of conformation and oligomeric state, confirming the structural information derived from the shorter construct. Currently, we are in the process of characterizing the thermodynamics and kinetics of Ca²⁺ binding in full-length EFD. Our results will provide insights into how Ca²⁺-induced conformational change is crucial for Duox activation.

Quincie Hayden

Appearances