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  • Structure of a domain-opene...
    Jameson, Geoffrey B.; Anderson, Bryan F.; Breyer, Wendy A.; Day, Catherine L.; Tweedie, John W.; Baker, Edward N.

    Acta crystallographica. Section D, Biological crystallography., June 2002, Volume: 58, Issue: 6-2
    Journal Article

    Human lactoferrin is an iron‐binding protein with a bilobal structure. Each lobe contains a high‐affinity binding site for a single Fe3+ ion and an associated ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N‐lobe half‐molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the ion at the iron‐binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular‐replacement solution Breyer et al. (1999), Acta Cryst. D55, 129–138, the structure has been refined at 3.0 Å resolution to an R factor of 13.9% (Rfree of 19.9%). Despite the moderate resolution, the high solvent content and non‐crystallographic symmetry contributed to electron‐density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion‐binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54°) closely matches that of the N‐lobe in full‐length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N‐lobe. Differences in the C‐­terminal portion of the N‐lobe (residues 321–332) for apoR121D relative to the closed wild‐type iron‐bound structure point to the importance of this region in stabilizing the open form.