| Summary: | <p>Abstract</p> <p>Background</p> <p>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<sup>st </sup>and 2<sup>nd </sup>EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>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 Ca<sup>2+ </sup>binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10<sup>5 </sup>and 5.0 × 10<sup>2 </sup>M<sup>-1 </sup>with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1<sup>st </sup>non-canonical EF-hand is largely enhanced by the binding of Ca<sup>2+ </sup>to the 2<sup>nd </sup>canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca<sup>2+ </sup>binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg<sup>2+</sup>-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1<sup>st </sup>non-canonical EF-hand of NOX5 has very weak Ca<sup>2+ </sup>binding affinity compared with the 2<sup>nd </sup>canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca<sup>2+ </sup>binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca<sup>2+ </sup>specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p>
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