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

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. 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.

When 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.

The 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.