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 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.

Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosa causes irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol

“…19 Additional structural investigations defined the crucial interface of the BfrB:Bfd complex 23 and analysis of this interface allowed us to demonstrate that mutation of two hot-spot residues in BfrB (L68A/E81A) is sufficient to block the BfrB:Bfd interaction and inhibit iron mobilization from BfrB. 24 To test these ideas in P. aeruginosa cells, and to investigate the consequences of removing BfrB, Bfd or the BfrB:Bfd interaction on iron homeostasis, we constructed mutant P. aeruginosa strains with unmarked, in-frame deletions of the bfrB (Δ bfrB ) or bfd (Δ bfd ) genes, and a strain with the bfrB gene encoding the L68A/E181A mutations ( bfrB (L68A/E81A)) (see Experimental). When wild type P. aeruginosa or mutant strains are cultured in iron sufficient media (PI media supplemented with 10 μM Fe), all the strains show nearly identical growth (Figure 2), although, as will be shown below, all three of the mutants grow significantly more slowly than wild type in iron-deplete conditions.…”

“…The K d values for the association between Bfd and the E81A and L68A mutants of BfrB are ~100-fold higher than wt BfrB, and the affinity of Bfd for the L68A/E81A double mutant of BfrB is undetectable. 24 In agreement, iron mobilization from the L68A/E81A BfrB mutant is completely inhibited in vitro , which indicates that the L68A/E81A mutation in BfrB blocks the BfrB:Bfd interaction and inhibits iron mobilization from BfrB. 24 …”

“…24 In agreement, iron mobilization from the L68A/E81A BfrB mutant is completely inhibited in vitro , which indicates that the L68A/E81A mutation in BfrB blocks the BfrB:Bfd interaction and inhibits iron mobilization from BfrB. 24 …”

“…Additional characterization of the BfrB:Bfd complex in solution showed that the 12 Bfd-binding sites on BfrB are equivalent and independent, and that the BfrB:Bfd interaction is characterized by a dissociation constant ( K d ) of approximately 3 μM. 24 Analysis of the interface showed that L68 and E81 in BfrB form a continuous set of interactions with M1, Y2 and L5 in Bfd (Figure 1e). The K d values for the association between Bfd and the E81A and L68A mutants of BfrB are ~100-fold higher than wt BfrB, and the affinity of Bfd for the L68A/E81A double mutant of BfrB is undetectable.…”

“…19 We cloned the genes, characterized the BfrB, Bfd and Fpr proteins biochemically and structurally, 20–22 and showed that Bfd, Fpr and NADPH are necessary and sufficient to mobilize iron from BfrB in vitro . 19,23,24 The X-ray crystal structure of the BfrB:Bfd complex 23 showed a biological assembly where 12 Bfd molecules bind a 24-mer BfrB; each Bfd occupies an identical site on BfrB, at the interface of a subunit dimer, above a heme molecule (Figure 1d). Additional characterization of the BfrB:Bfd complex in solution showed that the 12 Bfd-binding sites on BfrB are equivalent and independent, and that the BfrB:Bfd interaction is characterized by a dissociation constant ( K d ) of approximately 3 μM.…”