title: Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.
abstract: BACKGROUND
  Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear.
  
  
  RESULTS
  Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS.
  
  
  CONCLUSIONS
  Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding.
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan.
authors: Pei-Rung
authors: PR
authors: Wu
authors: Cheng-Chin
authors: CC
authors: Kuo
authors: Shaw-Fang
authors: SF
authors: Yet
authors: Jun-Yang
authors: JY
authors: Liou
authors: Kenneth K
authors: KK
authors: Wu
authors: Pei-Feng
authors: PF
authors: Chen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D004789:Enzyme Activation; D005415:Flavins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
chemical_list: D002147:Calmodulin; D005415:Flavins; D007527:Isoenzymes; D050505:Mutant Proteins; D010455:Peptides; D002956:Citrulline; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D014357:Trypsin; D002118:Calcium