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The three-dimensional structure of a form of human apolactoferrin, in which one lobe (the N-lobe) has an open conformation and the other lobe (the C-lobe) is closed, has been refined at 2.0 Å resolution. The refinement, by restrained least-squares methods, used synchrotron radiation X-ray diffraction data combined with a lower resolution diffractometer data set. The final refined model (5346 protein atoms from residues 1–691, two Cl− ions and 363 water molecules) gives a crystallographic R factor of 0.201 (Rfree = 0.286) for all 51305 reflections in the resolution range 10.0–2.0 Å. The conformational change in the N-lobe, which opens up the binding cleft, involves a 54° rotation of the N2 domain relative to the N1 domain. This also results in a small reorientation of the two lobes relative to one another with a further ∼730 Å2 of surface area being buried as the N2 domain contacts the C-lobe and the inter-lobe helix. These new contacts also involve the C-terminal helix and provide a mechanism through which the conformational and iron-binding status of the N-lobe can be signalled to the C-lobe. Surface-area calculations indicate a fine balance between open and closed forms of lactoferrin, which both have essentially the same solvent-accessible surface. Chloride ions are bound in the anion-binding sites of both lobes, emphasizing the functional significance of these sites. The closed configuration of the C-lobe, attributed in part to weak stabilization by crystal packing interactions, has important implications for lactoferrin dynamics. It shows that a stable closed structure, essentially identical to that of the iron-bound form, can be formed in the absence of iron binding.
