Structural characterization of the interaction of human lactoferrin with calmodulin.
Lactoferrin (Lf) is an 80 kDa, iron (Fe(3+))-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internal...
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| Format: | Article |
| Language: | English |
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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/PMC3516504?pdf=render |
| _version_ | 1818302588993404928 |
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| author | Jessica L Gifford Hiroaki Ishida Hans J Vogel |
| author_facet | Jessica L Gifford Hiroaki Ishida Hans J Vogel |
| author_sort | Jessica L Gifford |
| collection | DOAJ |
| description | Lactoferrin (Lf) is an 80 kDa, iron (Fe(3+))-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM), a ubiquitous, 17 kDa regulatory calcium (Ca(2+))-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa), TROSY-based NMR techniques were employed to structurally characterize Ca(2+)-CaM when bound to intact apo-Lf. Both CaM's backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca(2+)-CaM. Unlike the collapsed conformation through which Ca(2+)-CaM binds the CaM-binding domains of its classical targets, Ca(2+)-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca(2+)-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM's primary binding domain. Secondary interfaces play a key role in the target's response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes. |
| first_indexed | 2024-12-13T05:41:18Z |
| format | Article |
| id | doaj.art-9dc5ecd832ef44fc92fffc3e937962a6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T05:41:18Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-9dc5ecd832ef44fc92fffc3e937962a62022-12-21T23:57:47ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01712e5102610.1371/journal.pone.0051026Structural characterization of the interaction of human lactoferrin with calmodulin.Jessica L GiffordHiroaki IshidaHans J VogelLactoferrin (Lf) is an 80 kDa, iron (Fe(3+))-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM), a ubiquitous, 17 kDa regulatory calcium (Ca(2+))-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa), TROSY-based NMR techniques were employed to structurally characterize Ca(2+)-CaM when bound to intact apo-Lf. Both CaM's backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca(2+)-CaM. Unlike the collapsed conformation through which Ca(2+)-CaM binds the CaM-binding domains of its classical targets, Ca(2+)-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca(2+)-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM's primary binding domain. Secondary interfaces play a key role in the target's response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes.http://europepmc.org/articles/PMC3516504?pdf=render |
| spellingShingle | Jessica L Gifford Hiroaki Ishida Hans J Vogel Structural characterization of the interaction of human lactoferrin with calmodulin. PLoS ONE |
| title | Structural characterization of the interaction of human lactoferrin with calmodulin. |
| title_full | Structural characterization of the interaction of human lactoferrin with calmodulin. |
| title_fullStr | Structural characterization of the interaction of human lactoferrin with calmodulin. |
| title_full_unstemmed | Structural characterization of the interaction of human lactoferrin with calmodulin. |
| title_short | Structural characterization of the interaction of human lactoferrin with calmodulin. |
| title_sort | structural characterization of the interaction of human lactoferrin with calmodulin |
| url | http://europepmc.org/articles/PMC3516504?pdf=render |
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