Metal binding and conformational studies of the calcium binding domain of NADPH oxidase 5 reveal its similarity and difference to calmodulin

Abstract

The 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 actual hydrophobic exposure content. The heat capacity change (ΔCp) of Ca2+ binding for EFD is −24.1 cal/mol.K, while those of N-EFD and C-EFD are −56.3 and −41.6 cal/mol·K, respectively, indicating a significant hydrophobic exposure and polar burial. The latter was confirmed by limited trypsin digestion. The comparison of Nox5’s EFD to calmodulin, including homology modeling, was discussed in the report.Communicated by Ramaswamy H. Sarma