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PLoS One. 2012;7(6):e39851. doi: 10.1371/journal.pone.0039851. Epub 2012 Jun 29.

Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.

Author information

1
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan.

Abstract

BACKGROUND:

Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear.

METHODOLOGY/PRINCIPAL FINDINGS:

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.

PMID:
22768143
PMCID:
PMC3387242
DOI:
10.1371/journal.pone.0039851
[Indexed for MEDLINE]
Free PMC Article

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