Structure and domain dynamics of human lactoferrin in solution and the influence of Fe(III)-ion ligand binding

Abstract

BackgroundHuman lactoferrin is an iron-binding protein of the innate immune system consisting of two connected lobes, each with a binding site located in a cleft. The clefts in each lobe undergo a hinge movement from open to close when Fe3+ is present in the solution and can be bound. The binding mechanism was assumed to relate on thermal domain fluctuations of the cleft domains prior to binding. We used Small Angle Neutron Scattering and Neutron Spin Echo Spectroscopy to determine the lactoferrin structure and domain dynamics in solution.ResultsWhen Fe3+ is present in solution interparticle interactions change from repulsive to attractive in conjunction with emerging metas aggregates, which are not observed without Fe3+. The protein form factor shows the expected change due to lobe closing if Fe3+ is present. The dominating motions of internal domain dynamics with relaxation times in the 30–50 ns range show strong bending and stretching modes with a steric suppressed torsion, but are almost independent of the cleft conformation. Thermally driven cleft closing motions of relevant amplitude are not observed if the cleft is open.ConclusionThe Fe3+ binding mechanism is not related to thermal equilibrium fluctuations closing the cleft. A likely explanation may be that upon entering the cleft the iron ion first binds weakly which destabilizes and softens the hinge region and enables large fluctuations that then close the cleft resulting in the final formation of the stable iron binding site and, at the same time, stable closed conformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-016-0032-3) contains supplementary material, which is available to authorized users.

Dynamics of proteins in solution

“…Finally, A ( q ) for a set S of Brownian modes is given by (Biehl et al ., 2011) where the mode amplitudes a k depend on the friction constants and the temperature. A ( q ) as the q signature of internal motions has been successfully used to determine motional patterns occurring in proteins (Biehl et al ., 2008, 2011; Sill et al ., 2016).…”

Histidine-rich glycoprotein possesses antioxidant activity through self-oxidation and inhibition of hydroxyl radical production via chelating divalent metal ions in Fenton’s reaction

“…Fenton's reaction requires metal ions, for example, divalent iron ion, monovalent copper ion, and divalent cobalt ion, especially divalent iron ion is most important for this reaction [16][17][18]. Free-divalent iron ion is rigidly regulated by several proteins such as transferrin, lactoferrin, and ceruloplasmin in the bloodstream because its ion induces highly toxic hydroxyl radical production leading to tissue damage [38][39][40][41]. However, proteins binding to the divalent iron ion directly is not known in blood.…”

“…However, proteins binding to the divalent iron ion directly is not known in blood. Free divalent iron is generally oxidized by ceruloplasmin to trivalent iron, which binds to transferrin or lactoferrin and is metabolized [38,41]. However, direct binding to free divalent iron is thought to detoxify excess irons produced by tissue injury in septic conditions, more effectively.…”

Cancer Related Anemia: An Integrated Multitarget Approach and Lifestyle Interventions

“…An important role of LF in oxidation-reduction balance is based on its high antioxidant activity and inhibition of highly reactive oxidative agents. The ability of LF to bind the ferric ion (Fe 3+ ) [ 231 ] is twice as high as transferrin, the main plasma protein whose function, as previously described, is transporting iron in the bloodstream; both are part of the same family of proteins, named “transferrins”, capable of binding and transferring ions Fe 3+ ). Each LF molecule can bind two ferric ions to itself and based on this saturation it can exist in three distinct forms: apolactoferrin (iron-free), monoferric lactoferrin (linked to a single ferric ion), and ololactoferrin (which binds two ions to ferric itself) [ 231 ].…”

“…The ability of LF to bind the ferric ion (Fe 3+ ) [ 231 ] is twice as high as transferrin, the main plasma protein whose function, as previously described, is transporting iron in the bloodstream; both are part of the same family of proteins, named “transferrins”, capable of binding and transferring ions Fe 3+ ). Each LF molecule can bind two ferric ions to itself and based on this saturation it can exist in three distinct forms: apolactoferrin (iron-free), monoferric lactoferrin (linked to a single ferric ion), and ololactoferrin (which binds two ions to ferric itself) [ 231 ]. The oral integration of LF may be functional to maintain the oxide-reductive equilibrium, preventing iron from switching through Fenton reaction causing the synthesis of highly reactive oxidative compounds.…”