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

Abstract

BackgroundSuperoxide 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 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.ResultsThe 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 ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd 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 α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD.ConclusionsWe demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd 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

Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns

“…As a result, positive entropy is responsible for driving Mg 2+ binding to CML15. Interestingly, the N-terminal region of the Ca 2+ -binding domain of NADPH oxidase 5 (NCaBD of NOX5) generated a similar ITC isotherm to that of CML15 titrated with Ca 2+ (Linse and Chazin, 1995 ; Wei et al, 2012 ). ITC analysis of EF-hand I and EF-hand II of NCaBD suggests that EF-hand II has the higher affinity for Ca 2+ , triggering conformational changes that promote the binding of Ca 2+ to EF-hand I.…”

NOX5: Molecular biology and pathophysiology

“…The amount of Ca 2+ necessary to activate NOX5 fully is high, and accordingly, additional systems involving regulatory proteins are operational that increase sensitivity to Ca 2+ , thereby facilitating ROS generation at lower [Ca 2+ ] i . Hence, NOX5 can be activated directly by Ca 2+ or indirectly by interacting with other proteins and kinases, such as Ca 2+ ‐bound calmodulin or PKC (Wei et al., ).…”

Enzymatic regulation and functional relevance of NOX5

“…Calcium is essential for the activity of NOX5 and ROS are not synthesized to any appreciable degree in a calcium-free buffer or from truncated NOX5 enzymes lacking the N-terminal region 2-4 . The N- terminus of NOX5 contains 2 pairs of 4 EF hands that are distinguished in their relative affinity for calcium, the proximal N-terminal pair having a lower affinity to calcium than the distal pair 3, 5 . The current paradigm for activation is that upon elevation of calcium, the occupation of the EF hands triggers a conformational change in the N-terminus that promotes interaction with a C-terminal region thus facilitating electron flow from NADPH to oxygen bound to the heme moieties for the eventual production of superoxide and attendant reactive oxygen species.…”