Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

Background

Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^st and 2^nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.

Results

The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵ and 5.0 × 10² M^-1 with ??Hs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^st non-canonical EF-hand is largely enhanced by the binding of Ca²⁺ to the 2^nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its ??-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.

Conclusions

We demonstrated that the 1^st non-canonical EF-hand of NOX5 has very weak Ca²⁺ binding affinity compared with the 2^nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺ specific.

Graphical abstract