Structural Characterization of the Interaction of Human Lactoferrin with Calmodulin

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. © 2012 Gifford et al.