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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.
Highlights
Human 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
Structure: dependence of conformation on iron content The small angle neutron scattering (SANS) of proteins in solution depends on the protein form factor F(Q) and on the spatial arrangement between proteins comprised in the structure factor S(Q)
Apo-Lf showed to be consistent with a new homology model with both clefts open
Summary
Human 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. 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 close the cleft resulting in the final formation of the stable iron binding site and, at the same time, stable closed conformation. The high binding affinity is the cause for the main antibacterial activity as it removes the essential iron for bacteria growth beside a variety of other functions [3, 7]
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