Download MendeleyCrystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein.
Bruns, C.M., Anderson, D.S., Vaughan, K.G., Williams, P.A., Nowalk, A.J., McRee, D.E., Mietzner, T.A.(2001) Biochemistry 40: 15631-15637
- PubMed: 11747438
- DOI: https://doi.org/10.1021/bi0156759
- Primary Citation of Related Structures:
1D9V - PubMed Abstract:
The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.
Organizational Affiliation:
Department of Molecular Biology MB8, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
