Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+.
All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.
Ruan
J
J
Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021, USA.
Xie
Q w
Qw
Hutchinson
N
N
Cho
H
H
Wolfe
G C
GC
Nathan
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D004790:Enzyme Induction; D006801:Humans; D007668:Kidney / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D002118:Calcium