Crystallographic and Biochemical Analyses of the Metal-Free Haemophilus influenzae Fe3+-Binding Protein

详细信息    查看全文

• 作者：Christopher M. Bruns ; Damon S. Anderson ; Kevin G. Vaughan ; Pamela A. Williams ; Andrew J. Nowalk ; Duncan E. McRee ; and Timothy A. Mietzner
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：December 25, 2001
• 年：2001
• 卷：40
• 期：51
• 页码：15631 - 15637
• 全文大小：301K
• 年卷期：v.40,no.51(December 25, 2001)
• ISSN：1520-4995

文摘

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe³⁺-bindingprotein (hFbp) has been determined to 1.75 Å resolution. Information from this structure complementsthat derived from the holo structure with respect to the delineation of the process of iron binding andrelease. A 21 rotation separates the two structural domains when the apo form is compared with the holoconformer, indicating that upon release of iron, the protein undergoes a change in conformation by bendingabout the central -sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary bindingsite anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphatewith an affinity K_d of 2.3 × 10^-3 M. The presence of this ternary binding site anion appears to arrangethe C-terminal iron-binding residues conducive to complementary binding to Fe³⁺, while residues in theN-terminal binding domain must undergo induced fit to accommodate the Fe³⁺ ligand. These observationssuggest a binding process, the first step of which is the binding of a synergistic anion such as phosphateto 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 thesynergistic anion and this iron binding process results in an extremely high affinity of the Fe³⁺-bindingproteins for Fe³⁺ (nFbp K'_eff = 2.4 × 10¹⁸ M^-1). This high-affinity ligand binding process is uniqueamong the family of bacterial periplasmic binding proteins and has interesting implications in the mechanismof iron removal from the Fe³⁺-binding proteins during FbpABC-mediated iron transport across thecytoplasmic membrane.