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

Journal Article · · Biochemistry
 [1];  [1];  [1];  [1];  [2];  [1];  [1]
  1. Univ. of Kansas, Lawrence, KS (United States)
  2. Hauptman Woodward Medical Research Inst., Argonne, IL (United States). IMCA-CAT
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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. 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 (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 [LB68, EA81, and EA85 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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
National Center for Research Resources; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); National Science Foundation (NSF); Industrial Macromolecular Crystallography Association; USDOE Office of Science (SC); 2014 University of Kansas Strategic Grant
Grant/Contract Number:
5P20RR017708-10; 8 P20 GM 103420; AC02-06CH11357; MCB-1158469
OSTI ID:
1240165
Journal Information:
Biochemistry, Vol. 54, Issue 40; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 23 works
Citation information provided by
Web of Science

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Cited By (4)

Widespread distribution of encapsulin nanocompartments reveals functional diversity journal March 2017
Evidence that cyanobacterial Sll1217 functions analogously to PGRL1 in enhancing PGR5-dependent cyclic electron flow journal November 2019
Bacterioferritin: Structure, Dynamics, and Protein–Protein Interactions at Play in Iron Storage and Mobilization journal January 2017
Small Molecule Inhibitors of the BfrB–Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity journal May 2019

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Iron
Bioinorganic chemistry
Genetics
Molecules
Surface plasmon resonance