Identification of the binding site for the regulatory calcium-binding domain in the catalytic domain of NOX5.
NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.
Tirone
Fabiana
F
Departement of Biochemistry, University of Geneva, Geneva 4, Switzerland. fabiana.tirone@unige.ch
Radu
Laura
L
Craescu
Constantin T
CT
Cox
Jos A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D020832:EF Hand Motifs; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D014162:Transfection
D008565:Membrane Proteins; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D002118:Calcium