Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5).
NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
Department of Biochemistry, University of Geneva, CH-1211 Genève, Switzerland.
Botond
B
Bánfi
Fabiana
F
Tirone
Isabelle
I
Durussel
Judit
J
Knisz
Patryk
P
Moskwa
Gergely Zoltán
GZ
Molnár
Karl-Heinz
KH
Krause
Jos A
JA
Cox
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002474:Cell-Free System; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008555:Melitten / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013481:Superoxides / Q000378:metabolism
D008565:Membrane Proteins; D010446:Peptide Fragments; D013481:Superoxides; D008555:Melitten; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D002118:Calcium