title: Structural characterization of the interaction of human lactoferrin with calmodulin.
abstract: 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.
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Gifford
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ishida
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D002147:Calmodulin; D007781:Lactoferrin