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 (K_a’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⁺ (K_a = 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 (ΔC_p) 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

Keywords:

• EF-hand
• calcium binding
• NADPH Oxidase 5
• fluorescence
• isothermal titration calorimetry

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

We thank the internal financial supports from Southern Illinois University Edwardsville: Undergraduate Research and Creative Activities (URCA) to EF, EH, and LL and Graduate Students (RGGS) to EF, NK, and LL. This work is partly supported by the grant from the National Science Foundation grant CHE-1608484 to C.-C.W.