5d8o
1.90A resolution structure of BfrB (wild-type, C2221 form) from Pseudomonas aeruginosa
Structural highlights
FunctionBFRB_PSEAE The major iron-storage protein, part of the heterooligomeric bacterioferritin (BFR) complex. The ferroxidase center binds Fe(2+), oxidizes it using dioxygen to Fe(3+), and participates in the subsequent Fe(3+) oxide mineral core formation within the central cavity of the BFR protein shell. Can store up to 600 iron atoms per bacterioferritin protein molecule (PubMed:19575528, PubMed:20067302, PubMed:25640193, PubMed:26368531). In iron-sufficient conditions (10 uM Fe(2+)) iron accumulates in BFR until about 12 hours, when it starts to deplete; stored iron is no longer detectable by 24 hours growth, iron is mobilized from the BFR as levels drop in the growth media (PubMed:28318006). Iron release from the BFR requires ferredoxin NADP reductase (FPR) and bacterioferritin-associated ferredoxin (Bfd) (PubMed:19575528, PubMed:22812654, PubMed:26368531). Reduction of the BfrB heme group occurs in the presence of Bfd, strongly suggesting that the BfrB-Bfd complex allows heme to mediate electron transfer from FPR to the Fe(3+) iron core in the BFR shell prior to its release as Fe(2+) (PubMed:19575528, PubMed:22812654, PubMed:26368531).[1] [2] [3] [4] [5] [6] Publication Abstract from PubMedMobilization 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 P. 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., Wang, Y., Lovell, S., Kumar, R., Ruvinsky, A. M., Battaile, K. P., Vakser, I. A., and Rivera, M. J. Am. Chem. Soc. (2012), 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 (Kd) of approximately 3 muM. 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 [L_B^68, E_A^81 and E_A^85 in BfrB (superscript for residue number and subscript for chain) and Y2 and L5 in Bfd], which 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. Characterization of the Bacterioferritin/Bacterioferritin Associated Ferredoxin (BfrB:Bfd) Protein-Protein Interaction in Solution and Determination of Binding Energy Hot Spots.,Wang Y, Yao H, Cheng Y, Lovell SW, Battaile KP, Middaugh CR, Rivera M Biochemistry. 2015 Sep 28. PMID:26368531[7] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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