Characterization of the Bacterioferritin/Bacterioferritin Associated Ferredoxin Protein-Protein Interaction in Solution and Determination of Binding Energy Hot Spots

摘要

Mobilization of iron stored in the interior cavity of BfrB requires electron transfer from the [2Fe-2S] cluster in Bfd to the core iron in BfrB. A crystal structure of the Pseudomonas aeruginosa BfrB:Bfd complex revealed that BfrB can bind up to 12 Bfd molecules at 12 structurally identical binding sites, placing the [2Fe-2S] cluster of each Bfd immediately above a heme group in BfrB [Yao, H., et al. (2012) J. Am. Chem. Soc., 134, 13470-13481]. We report here a study aimed at characterizing the strength of the P. aeruginosa BfrB:Bfd association using surface plasmon resonance and isothermal titration calorimetry as well as determining the binding energy hot spots at the protein-protein interaction interface. The results show that the 12 Bfd-binding sites on BfrB are equivalent and independent and that the protein-protein association at each of these sites is driven entropically and is characterized by a dissociation constant (K-d) of approximately 3 mu M. Determination of the binding energy hot spots was carried out by replacing certain residues that comprise the protein-protein interface with alanine and by evaluating the effect of the mutation on K-d and on the efficiency of core iron mobilization from BfrB. The results identified hot spot residues in both proteins [L-B(68), E-A(81), and E-A(85) in BfrB (superscript for residue number and subscript for chain) and Y-2 and L-5 in Bfd] that network at the interface to produce a highly complementary hot region for the interaction. The hot spot residues are conserved in the amino acid sequences of Bfr and Bfd proteins from a number of Gram-negative pathogens, indicating that the BfrB:Bfd interaction is of widespread significance in bacterial iron metabolism.