Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.
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...
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| Format: | Article |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3387242?pdf=render |
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| author | Pei-Rung Wu Cheng-Chin Kuo Shaw-Fang Yet Jun-Yang Liou Kenneth K Wu Pei-Feng Chen |
| author_facet | Pei-Rung Wu Cheng-Chin Kuo Shaw-Fang Yet Jun-Yang Liou Kenneth K Wu Pei-Feng Chen |
| author_sort | Pei-Rung Wu |
| collection | DOAJ |
| description | 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. |
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| id | doaj.art-c326b9405ba34811bce4428b7ab07013 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T07:21:58Z |
| publishDate | 2012-01-01 |
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| series | PLoS ONE |
| spelling | doaj.art-c326b9405ba34811bce4428b7ab070132022-12-22T02:06:08ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0176e3985110.1371/journal.pone.0039851Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.Pei-Rung WuCheng-Chin KuoShaw-Fang YetJun-Yang LiouKenneth K WuPei-Feng ChenBACKGROUND: 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.http://europepmc.org/articles/PMC3387242?pdf=render |
| spellingShingle | Pei-Rung Wu Cheng-Chin Kuo Shaw-Fang Yet Jun-Yang Liou Kenneth K Wu Pei-Feng Chen Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. PLoS ONE |
| title | Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. |
| title_full | Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. |
| title_fullStr | Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. |
| title_full_unstemmed | Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. |
| title_short | Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation. |
| title_sort | lobe specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation |
| url | http://europepmc.org/articles/PMC3387242?pdf=render |
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