title: Characterization of the Bacterioferritin/Bacterioferritin Associated Ferredoxin Protein-Protein Interaction in Solution and Determination of Binding Energy Hot Spots.
abstract: 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 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 (Kd) of approximately 3 μM. 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 Kd and on the efficiency of core iron mobilization from BfrB. The results identified hot spot residues in both proteins [LB 68, EA 81, and EA 85 in BfrB (superscript for residue number and subscript for chain) and Y2 and L5 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.
authors.affiliation: Department of Chemistry, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Room 220E, Lawrence, Kansas 66047, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Room 220E, Lawrence, Kansas 66047, United States.
authors.forename: Huili
authors.initials: H
authors.lastname: Yao
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Room 320G, Lawrence, Kansas 66047, United States.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas , 2034 Becker Drive, Lawrence, Kansas 66047, United States.
authors.forename: Scott
authors.initials: S
authors.lastname: Lovell
authors.affiliation: IMCA-CAT, Hauptman Woodward Medical Research Institute , 9700 South Cass Avenue, Building 435A, Argonne, Illinois 60439, United States.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Battaile
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Room 320G, Lawrence, Kansas 66047, United States.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Midaugh
authors.affiliation: Department of Chemistry, University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive, Room 220E, Lawrence, Kansas 66047, United States.
authors.forename: Mario
authors.initials: M
authors.lastname: Rivera
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D060066:Protein Interaction Maps; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D005288:Ferredoxins; D005293:Ferritins; C018032:bacterioferritin