Abstract
Lactoferrin (Lf) is an 80 kDa, iron (Fe3+)-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 (Ca2+)-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 Ca2+-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 Ca2+-CaM. Unlike the collapsed conformation through which Ca2+-CaM binds the CaM-binding domains of its classical targets, Ca2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca2+-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.
Highlights
In humans, lactoferrin (Lf) is a cell-secreted, 80 kDa host defense glycoprotein with antibacterial, antiviral, antifungal, antitumor, and anti-inflammatory activities [1,2]
Lf is secreted in its Fe3+-free form by epithelial cells into most exocrine fluids, or alternatively, is released at a very high concentration from the secondary granules of activated neutrophils recruited to sites of inflammation
The interaction between apo- or Fe3+-Lf and Ca2+-CaM was studied through fluorescence spectroscopy by measuring changes in the fluorescence emission spectrum of dansylated CaM
Summary
Lactoferrin (Lf) is a cell-secreted, 80 kDa host defense glycoprotein with antibacterial, antiviral, antifungal, antitumor, and anti-inflammatory activities [1,2]. Lf is secreted in its Fe3+-free (apo) form by epithelial cells into most exocrine fluids, or alternatively, is released at a very high concentration from the secondary granules of activated neutrophils recruited to sites of inflammation. Fe3+-independent antimicrobial activities have been ascribed to Lf. Fe3+-independent antimicrobial activities have been ascribed to Lf These activities are linked to the ability of this highly cationic (pI , 9) protein, in particular its N-terminal lactoferricin domain, to interact with negatively charged molecular and cellular components of both host cells and pathogens [5]
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