Metal Binding and Conformational Studies of the Calcium Binding Domain of NADPH Oxidase 5 Reveal its Similarity and Difference to Calmodulin

AbstractThe superoxide-generating activity of Nox5 is regulated by Ca2+ flux, primarily through its self-contained calcium binding domain (EFD). Upon Ca2+ binding, Nox5’s EFD undergoes a conformational change that exposes its buried hydrophobic residues. Previously, we determined the Ca2+ binding constants of the N-terminal half domain (N-EFD). Here we performed a similar characterization with its C-terminal lobe (C-EFD). Our studies revealed that the binding affinities (Ka’s) of the EFD are in the range of 108-105 M-1 with a strong Ca2+ binding that occurs in the C-EFD. The 3rd Ca2+ binding site also binds Mg2+ (Ka = 4.53 × 103 M-1), where its high Ca2+ binding affinity becomes moderate in cellular conditions. The essential hydrophobic exposure upon metal binding was assessed with the analysis of the 1-anilino-8-naphthalene sulfonate (ANS) interaction via fluorescence and calorimetry. While the ANS fluorescence and binding studies agree with each other in general, the results do not correlate to the actu...